The corpuscles of Stannius contain essential elements of the ...

The corpuscles of Stannius contain essential elements of the renin- angiotensin systern for the regulation of blood flow in freshwater North American eels, Anguilla rostrata Lesueur.

Donald H. Zhang

A thesis submitted in confomity with the requirements for the degree of Master of Science

Graduate Department of Zoology University of Toronto

O Copyright by Donald H. Zhang (1999)

National Library 1+1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

395 Wellington Street 395. rue Weflhgton OaawaON K 1 A M OttawaON K 1 A W Canada Canada

The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sel1 copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantid extracts fkom it may be printed or otheMrise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni Ia thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

ABSTRACT

The corpuscles of Stannius contain essential elements of the renin-

angiotensin system for the regulation of blood flow in freshwater North

American eels, Anguilla rostrata Lesueur.

Master of Science, 1999

Donald H. Zhang

Department of Zoology. University of Toronto.

These experiments examine how elements of the renin-angiotensin

system (RAS) may modulate dorsal aortic blood flow (DABF) and caudal venous

blood flow (CVBF) in a mode[ teleost fish. the freshwater North American eel

(Anguilla rostrata Lesueur). The experiments also examine the theory that the

corpuscles of Stannius (CS) are part of the RAS in freshwater eels and that they

may secrete renin. Blood Row rates were measured in the dorsal aorta (DA) and

the caudal vein (CV) of free-swimming, conscious freshwater (FW) eels. using ,

surgically implanted Doppler flow probes. DABF and CVBF increased in (FW)

eels in a dose-dependent manner following i.v. injections of [~sn' . val5, ~ 1 ~ 4 - Angiotensin I (ANG 1). [ ~ sn ' . ~al7-~ngiotensin II (ANG II) and al^]-~ngiotensin

III (ANG III). Doses were given in 5 ng increments, ranged from 5 -50 ng.kg bw?

A minimum effective dose for ANG I and ANG II was 5 ng-kg bw-'; for ANG 111. 10

ng-kg bw? In most cases. both DABF and CVBF flow increased during the first 2

minutes and remained elevated for 20-50 minutes (min) before decreasing to the

pre-injection rates. [ ~ a r ' . V~IY-ANG II (Sarile). the AT1 And AT2 receptor

antagonist. completely blocked the increases in DABF and CVBF in responses to

ANG II. Losartan, the mammalian AT1 antagonist, and PD 123319. the

rnamrnalian AT2 antagonist. both blocked partially. the increased DABF and

CVBF which followed injections of ANG II. Failure of both Losartan and

PD123319 to completely abolish the flow response to ANG II and CS-EXT

suggest strongly that the blood flow responses to angiotensins is governed by

more than one Angiotensin l l receptor subtype in the eel-

An i.v. injection of an extract of 2.5 mg fresh corpuscles of Stannius (CS-

EXT). presumed to contain renin. was followed by an immediate and sustained

elevation in DABF and CVBF which lasted for approximately 30 min. A similar

increase in DABF and CVBF followed the i.v. injection of 150 ng-kg bw" of

human renin substrate (hRS). An extract of posterior kidney (PK-EXT) had no

effect on either DABF or CVBF. Flow responses to CS-EXT and hRS were

blocked completely by a prior i.v. injection of 1 mg-kg bw-' of the mammalian

renin inhibitor, Pepstatin A. Pepstatin A did not block the flow responses to either

ANG I or ANG II. lntravenous injection of 1 rng-kg bw*' of Captopril. the

mammalian angiotensin-converting enzyme (ACE) inhibitor, completely abolished

the DABF and CVBF responses to CS-EXT, hRS and ANG 1, while the flow

responses to ANG II was unaffected.

These findings in conjunction with other works on freshwater eels lead to

the conclusion that angiotensins act centrally or via catecholamine release from

the peripheral sympathetic nervous system or chromaffin cells of the anterior

head kidney to increase cardiac output (CO). Moreover, my experiments have

shown that eel CS contain renin or a renin-like substance that is a cornponent of

an eel RAS.

I thank my supervisor, Dr. D. G. Butler. for the kindness and the patience

he has shown me throughout the course of my training. His enthusiasm and

cornmitment to the art and discipline of research has been both inspiring and

edifying. I will greatly miss our long and scholarly discussions that have left

indelible imprints on my growing awareness of the world.

To Alia Cadinouche. Gavin Oudit, Sunny Pak. Colleen Roe and Roharn

Zandevakili. I extend to you my sincerest gratitude for your friendship and

support. I am especially indebted to Sunny. for her tireless and invaluable

editorial feedback. to Colleen. for introducing me to the Doppler technique. and to

Roham. for his vast cornputer expertise. Many thanks to Gavin and Alia. for

making me feel welcomed and accepted in the lab.

I further extend my appreciation to Dr. R. Stephenson for the use of his

Doppler Flowmeter, and to Dr. D. Jackson and Kristie Ciruna for their statistical

counseling. I am also indebted to Diana Powell and Rosanna Soo for their

assistance in revising my thesis. Many thanks to Eric Lin, Sonia McKenzie, Kim

Gallant. Terry Hill. Elizabeth Tudor-Mulroney and Steve Smith for making my

graduate experience a more enjoyable one.

To my family. words cannot express the full extent of my gratitude for your

unfaltering love and support. Thank you for tolerating me during the often difficult

and circuitous course of my training.

TABLE OF CONTENTS

abstract

Acknowledgments

Table of Contents

List of Figures

List of Tables

List of Abbreviations

INTRODUCTION

The Renin-Angiotensin System (RAS)

(A) The RAS in mammals (B) Evolution of the RAS (C) The RAS in fishes

i. Cyclostomes i i . Holocephalans iii. Elasmobranchs iv. Sarcopterygians and ancient bony fishes v. Modern bony fishes

The corpuscles of Stannius (CS) of Teieost Fishes

(A) The RAS and the CS (B) Distribution of the CS .

(C) Vascularization of the CS (D) Innervation of the CS (E) Cell types of the CS

Pharmacological Inhibition of the RAS

(A) lnhibition of renin (B) lnhibition of angiotensin-converting enzyme (ACE) (C) lnhibition of angiotensin II receptors

Objective

MATERIALS AND METHOD

Experimental animals

Experimental set-up and surgical procedures

(A) Insertion of Doppler flow probes (8) Collecting the corpuscles of Stannius and posterior kidney tissue (C) Insertion of dnig delivery catheter (D) Calibration of Doppler flow probes

Drugs and peptides

Blood fiow experiments: Experimental protocol

Series 1: Response to components of the RAS Series II: Response to RAS antagonists

RESULTS

Cali bration Cuwes and velocity profiles

(A) ANG 1. ANG II or ANG III and blood flow (B) Tissue extracts and blood flow (C) Pepstatin A and the blood flow responses to ANG 1, ANG II, hRS or CS-EXÏ (D) Captopril and the blood flow responses to ANG 1, ANG II, hRS or CS-EXT (E) Sarile and the blood flow response to ANG II or CS-EXT (F) Losartan and the blood flow responses to ANG II or CS-EXT (G) PD 12331 9 and the blood flow responses to ANG II or CS-EXT

DISCUSSION

The effects of ANG I and ANG II on blood flow The relationship between CO, DABP and DABF and CVBF The effects of ANG III on blood flow Evidence for a renin or renin-like enzyme in the CS Inhibition of putative renin in eel CS-EXT and of hRS by pepstatin A

Blood flow responses to putative renin in eel CS-EXT and to hRS

(A) Inhibition of the endogenous conversion of ANG I to ANG II by Captopril (8) Effects of the ANG II receptor antagonist Sarile (C) Effects of subtype specific receptor antagonists Losartan and PD12331 9

SUMMARY

REFERENCES 102

vii

LIST OF FlGURES

Figure 1. Caudal venous ( open circle ) and dorsal aortic ( closed circle )

calibration curves ( n = 3 ) 35

Figure 2. Caudal venous blood flow ( CVBF ) ( open column ) and dorsal aortic

blood flow ( DABF ) ( closed culurnn ) response to graded i-v.

injections of ANG 1. ANG II and ANG III ( n = 5 ) 37

Figure 3. CVBF ( open column ) and DABF ( closed column ) response to graded

i.v. injections of hRS and CS-EXT ( n = 5 ) 40

Figure 4. Effect of the renin antagonist Pepstatin A on the temporal changes in

CVBF ( open column ) and DABF ( closed column ) in response to i.v.

injections of ANG I ( n = 5 ) 43



injections of ANG II ( n = 5 ) 45



injections of hRS ( n = 5 ) 47



injections of CS-EXT ( n = 5 ) 49

Figure 8. Effect of the ACE antagonist Captopril on the temporal changes in

CVBF ( open column ) and DABF ( closed column ) in responseto i-v.

injections of ANG I ( n = 5 ) 52

Figure 9. Effect of the ACE antagonist Captoprïl on the temporal changes in


injections of ANG II ( n = 5 ) 54

Figure 10. Effect of the AC€ antagonist Captopnl on the temporal changes in

CVBF ( open column ) and DABF ( closed column ) in response to i-v.

injections of hRS ( n = 5 ) 56

Figure 1 1. Effect of the ACE antagonist Captopril on the temporal changes in


injections of CS-EXT ( n = 5 ) 58

Figure 12. Effect of the ANG II receptor antagonist Sarile on the temporal

changes in CVBF ( open column ) and DABF ( closed column ) in

response to i.v. injections of ANG II ( n = 5 ) 61

Figure 13. Effect of the ANG II receptor antagonist Sarile on the temporal

changes in CVBF ( open colurnn ) and DABF ( closed column ) in

response to i-v. injections of CS-EXT ( n = 5 ) 63

Figure 14. Effect of the AT, receptor antagonist Losartan on the temporal



Figure 15. Effect of the AT, receptor antagonist Losartan on the temporal


response to i.v. injections of CS-EXT ( n = 5 ) 68

Figure 16. Effect of the AT2 receptor antagonist P D ~ 2331 9 on the temporal

changes in CVBF ( open colurnn ) and DABF ( closed column ) in


Figure 17

Figure 18

Effect of the AT2 receptor antagonist PD1 2331 9 on the temporal


response to i-v. injections of CS-EXT ( n = 5 ) 73

Temporal changes in CO (A) [ control ( n = 8. closed circle ) and

atropine treated ( n = 6. open circle ) 1, mean PDA ( B; n = 6 ), and

Rsys ( C; n = 6 ) after i.v. injection of ANG II ( Adapted with

permission from Oudit and Butler. 1995 ) 80

Figure 19. Stepwise pharmacological inhibition of the blood flow responses to

peptide components of the eel RAS. including the possible role of the

CS. 98

LIST OF TABLES

Table 1. Primary structure of angiotensin I from select vertebrate species 9

Cc

AC€ ANG I ANG II ANG III ANOVA Asn A ~ P OC

cm CO CRAS CS CS-EXT cv CVBF DA DABF EGM 9 G ~ Y hRS i.d. i.v. i.m. JG JGA KDa Kg bw Khz 1 Ci9 MD mg min ml mm MS222 MW n n g NaCl

Alpha Angiotensin converting enzyme Angiotensin I Angiotensin II Angiotensin III Analysis of variance Asparagine As partate Degree centigrade Centimeter Cardiac output Corpuscular renin-angiotensin system Corpuscles of Stannius Corpuscles of Stannius extract Caudal vein Caudal venous blood flow Dorsal aorta Dorsal aortic blood flow Extraglomenilar mesangial cells Gram Glycine Human renin-su bstrate lnner diameter lntravenous intramuscular Juxtaglomerular cells Juxtaglomerular apparatus Kilodalton Kilogram body weight Kilohertz Litre Microgram Macula densa Miligram Minute Mililitre Miiirnetre Methane tricanesulphonate Molecular weight Sample size Nanogram Sodium chloride

xii

0-d. PS P PDA PE PK-EXT r RAS RS rrn SEM Val VR

Outer diameter Picogram Attained significant ievel Dorsal aortic blood pressure Polyeth ylene Posterior kidney extract Correlation coefficient Renin-angiotensin system Renin substrate repeated measures Standard error of the mean Valine Venous retum

INTRODUCTION

The corpuscles of Stannius (CS) are putative endocrine glands that may regulate

cardiovascular function in teleostean fishes. in concert with the Renin-angiotensin

system (RAS). Chester-Jones first described a rapid decline in blood pressure in

European eels. Anguilla anguiiia. following extirpation of the paired glands (Chester-

Jones et ai. 1966). Subsequently, he showed that extracts of the CS contain a powerful

renin-like pressor substance. in the anaesthetized rat bioassay. More recently, the CS

have been shown to contain angiotensin I (ANG 1) and angiotensin II (ANG II). in a wide

variety of teleost species (Hasegawa et ai. 19Wa. Takemoto et al. 1983, Yamada &

Kobayashi 1987). The present study was undertaken to determine whether the CS in a

model teleost fish. the North American eel, Anguilla rostrata Lesueur, contain

physiologicaily significant renin-like activity. In these experirnents, the effects of an

extract of CS on the regulation of blood flow were investigated in conscious eels, with

the aid of chemical antagonists of the RAS.

(A) The RAS in marnmals

Brown and Séquard have been credited with the discovery of the RAS over a century

ago, in 1892. Since its discovery, the vast majority of research on the RAS has been

limited to studies on rnarnmals. In marnmals. the RAS is an intricate enzymatic cascade

comprising 7 essential elements: 1) The enzyme renin. an aspartyl released by the

juxtaglomerular cells (JG) of renal afferent and efferent arterioles. 2) the protein

angiotensinogen. an U, globulin renin-substrate produced by the liver, 3) angiotensin-

converting enzyme (ACE). a dipeptidyl carboxypeptidase occurring mostly in the lung

endothelium. and 4) angiotensinases that degrade the active hormones of the RAS into

smaller, predorninately inactive peptides. The active products of the RAS consist of

angiotensin (ANG) peptides of varying lengths (7-10 amino acids), including 5) the ANG

1 decapeptide. 6) the ANG II octapeptide and 7) the ANG III heptapeptide. ANG II is

considered the most potent of these peptides, in terrns of its pressor effects and its

primary function in the regulation of body fl uid homeostasis .

Traditionally. the juxtaglomenilar apparatus (JGA) was considered essential for

the proper functioning of the RAS. Yet only birds and marnmals have intact JGA's. even

though species from every vertebrate class are dependent on the RAS (Nishimura

1980a. b). In birds and marnmals, juxtaglomenilar (JG) cells containing renin and

prorenin granules are found in both the afferent and the efferent arterioles of the unit

nephron. Apposed to one side of the afferent arteriole are specialized epithelial cells of

the renal tubule called macula densa cells (MD). The MD act as sodium sensors which

respond to drops in tubular lumina1 sodium concentration, by stimulating renin secretion

from JG cells. Accessory cells associated with the JGA include extraglomenilar

mesangial cells (EGM) which are interspersed between the MD and the glomerulus. A

second cell type. known as peripolar cells (Ryan et al. 1979). can be found encircling

the origin of the glomerular tuft, although. their function is hitherto unknown.

(B) Evoiution of the RAS

Comparative studies on the RAS began in the late 1960s. and it became rapidly

apparent that all vertebrate classes have a RAS despite an incomplete or altogether

absent JGA (Edwards 1940, Capelli et al.1970, Sokabe et a1.1969). Studies on the RAS

of fishes. amphibians and reptiles necessarily focus on the physiological and the

biochemical features of the RAS. rather than on the presence of the JGA (Nishimura et

al. 1973). Incubation of renal extract with homologous plasma produces angiotensins in

select species frorn every vertebrate class. from primitive jawless fishes to mammals. In

addition, incubation of renal extracts with homologous plasma. under the condition of

angiotensinase inhibition, produce ANG Il-like pressor substance in virtually every

animal mode! examined (see review by Sokabe 8 Ogawa 1974).

A preponderance of research strongly suggests that the RAS is an evolutionanly

ancient system whose ancestry may be traced as far back as invertebrates. ANG Il-like

substances have been isolated in the nervous systems of the leech, Theromyzon

tessulatum, (Verger-Bocquet et al. 1992, Salzet et al. 1992. 1993). the earthworm,

Eisenia foetida (Kobayashi 8 Takei 1996a) and the amphioxus. Branchiostoma belcheri

(Uemura et al. 1994). In invertebrates, ANG Il-like substances are thought to subserve

a neuromodulatory function, whereas in vertebrates, it plays a pivotal role in

cardiovascular regulation as welt as fluid and electrolyte homeostasis. Hence, an

evolutionary transition in the function of ANG II may have occurred from that of a

neuromodulator to a regulator of cardiovascular function.

The presence of cells with renin-containing granules that have similar staining

and chernical properties to their mammalian counterpart, is an a prion requirement for

the existence of a RAS within a vertebrate class. On the basis of this criteria, the RAS

seems to have evolved first in fishes (Nishimura 1987). Primitive bony fishes. teleosts,

lungfishes, amphibians, and reptiles al1 have granulated cells resembling mammalian

JG cells in the small arteries and the artenoles of the kidney. In fishes, up to 6 different

types of JG cell distributions have been described along the renal artery and arterioles

(Krishnamurthy & Bern 1969). Vasopressor substances with similar biological properties

to mammalian angiotensins have been formed from kidney extracts of lower vertebrates

including primitive bony fishes, modern bony fis hes. amphibians, reptiles. and birds

(Sokabe et al. 1969: Nishimura et al. 1973).

(C) The RAS in Fishes

i. Cyclostomes Studies on the cyclostome RAS have focused on 3 species: the

lamprey. Lampetra japonica. the hagfish. and the myxinoids Paramyxine atami and

Myxine glutinosa (Nishimura et al. 1970. Oguri et al. 1970). Cyclostomes do not appear

to have a RAS, since they Jack granulated JG cells (Sokabe et al. 1969). Nevertheless,

exogenous ANG II can stimulate vasoconstriction. drinking behaviour and

mineralocorticoid release in the hagfish (Carroll 8 Opdyke 1982). Similarly. renal

extracts of the lamprey. incubated with canine renin-substrate, produce ANG Il-like

pressor substances (Henderson et al. 1981). Most recently, Takei and Rankin have

isolated ANG I from the lamprey (Kobayashi & Takei 1996b). In Iight of these new

findings. more detailed studies on the histological basis of the RAS in cyclostomes may

be warranted.

ii. Holocephalans There is a small number of studies on the RAS in holocephalans. In

the rabbitfkh. Chimaera monstrosa (Oguri 1 978. 1980) and the ratfish. Hydolagus

colloeo (Nishimura et al. 1973. Oguri 1978). cells similar to rnammalian JG cells have

been detected in the afferent arteriole. near the glomenilus. Incubation of ratfish kidney

extract with hornologous plasma produced a pressor substance sirnilar to ANG Il

(Nishimura 1985). however, the existence of a JGA is doubtful since both species lack

MD-

iii. Elasmobranchs Early histological studies failed to detect JG cells in

elasmobranchs (see reviews by Sokabe 8 Ogawa 1974. Wilson 1984. Nishimura 1985).

Of the species examined. renin could not be detected in kidney extracts (Bean, 1942.

Nishimura et al. 1970, Nishimura, 1985). However, these studies relied on the old

Bowie's staining rnethod. and with the development of the toludine-blue staining

technique as well as more sensitive assays for renin, the presence of the RAS in

elasrnobranches has since been clearly validated. Granulated JG cells have been

identified in 4 species of elasmobranchs (Lacy & Reale 1990) along with the MD. EGM

and the peripolar cells.

ANG II has also been detected in the brain. kidney, pituitary and rectal glands of

the nurse shark. Gynclymostoma cirratum. In the spiny dogfish. Squalus acanthias.

exogenous ANG I and ANG II elicits pronounced pressor responses. Furthermore, the

effect of ANG 1 was blocked by the ACE inhibitor, SQ 20881 (Opdyke 8 Holcombe 1976

) Direct intravenous injection of dogfish. Scyliohinus canicula. renal extract or the

injection of renal extract incubated with rat renin substrate, produced an ANG Il-like

pressor effect in nephrectomized rats (Henderson et al. 1981). In the dogfish and spiny

dogfish, exogenous ANG II elicits both pressor and dipsogenic responses and

stimulates the release of mineralocorticoids (Hazon 8 Henderson 1985. Hazon et al.

1989, O'Toole et al. 1990). ANG II stimulates the secretion of 1 a-

hydroxycorticosterone from the isolated and perfused intenenal gland of the dogfish

(Armour et ai. 1993). Plasma levels of ANG II in the nurse shark range between 30 to

80pglml (Galli-Phillips, 1991). and these levels increase significantly following

hemorrhage or transfer from freshwater to 25% seawater.

Most recently. ANG I frorn the dogfish. Tnakis scyllia, has been isolated and fully

sequenced with startling results(Takei et al. 1993b). As in mammals, isoleucine occurs

in the fifth position of the amino acid sequence of dogfish ANG I . a finding which may

have important phylogenetic implications. In addition. dogfish is unique among

vertebrates since its ANG I contains proline occurs in the third position. instead of

valine.

iv. Sarcopterygians and ancient bony fishes In the kidneys of crossopterygians,

coelacanthodi and Latimena chalumnae. Bowie stain failed to detect JG granules

(Nishimura & Ogawa. 1973). however. renin-activity was present in their kidney extracts

(Nishimura et al. 1973). In dipnoan fishes. granulated cells resembling JG cells have

been identified in the tunica media of renal arteries. Renal renin activity has been found

in the kidney extracts of the dipnoan. Protopterus aethiopicus. and Lepidosiren

paradoxa (Ogawa et al. 1972. Nishimura et al. 1973). and plasma renin activity has

been detected in a third species. Neoceratodus fosten (Blair-West et al. 1977).

To date, studies on the holostean RAS have been limited to only two species;

the bowfin, Amia calva and the longnosed garpike. Lepisosteus osseus. Although

granulated JG cells have only been identified in the garpike (Ogawa et al. 1972.

Nishimura et al. 1973). kidneys of both species contain significant renin-activity

(Nishimura et a/. 1973). In the bowfin, expenmental evidence suggests that the

cardiovascular system is regulated by a RAS (Butler et al. 1995). Exogenous ANG II

was found to increase arterial blood pressure, a response that c m be blocked by the

ACE inhibitor Captopril. In the bowfin, ANG II and its receptors may have evolved .. somewhat independently since exogenous eel-[Asn', Val5. GlflANG II and bowfin-

[Asp', Val5. Gly5]-ANG II have identical pressor effects in the bowfinuakei et al. 1998).

In 4 species of chondrosteans, the Bowie's stain method failed to detect

granulated JG cells (Ogawa et al. 1972. Nishimura et aL 1973, Krishnamurthy & B e n

1969). Nevertheless, kidney extracts from Polypterus senegalus show renin activity,

suggesting the presence of a chondrostean RAS (Nishimura et al. 1973). Of the

sarcopterygian fishes examined thus far. there is no evidence of a JGA. Stnictures-

relevant to the JGA, including the MD and the EGM have not been detected (Nishimura

et al. 1973, Sokabe & Ogawa 1974. Lagios 1974).

v. Modern bony fishes (Teleosteans) The teleostean RAS is the most extensively

studied among fishes. Granulated JG cells have been observed in numerous species of

teleosts using the Bowie's stain method and the periodic acid Schiff stain method

(Sokabe & Ogawa, 1974, Oguri 8 Sokabe, 1968, Krishnamurthy 8 Bern, 1969). In

teleosts. there is no evidence of the MD (Krishnamuithy & Bem, 1969). or EGM cells in

teleosts (Sokabe 8 Ogawa, 1974. Ogawa & Oguri 1978).

50th plasma renin activity and renin-substrate have been detected in the

toadfish. Opsanus tau (Nishimura, 1980). Such studies provide strong indirect evidence

for a renin or a renin-like enzyme in teleosts. In the European eel. A. anguilla. and the

rainbow trout, Oncorhynchus mykiss. exogenous ANG II increases plasma cortisol

levels (8omraja et al. 1973. Arnold-Reed 8 Balment 1994). Exogenous ANG II can also

increase water intake in teleosts (Perrott 8 Balment 1985. Perott et al. 1992, Hirano et

al. 1978. Takei et al. 1979b. Hirano & Hasegawa 1984).

ANG I has been sequenced in 4 species of teleosts: the aglomerular goosefish,

Lophius litulon (Hayashi et al. 1978). the chum salmon. Oncorhynchus keta (Takemoto

et al. 1983). the Japanese eel. A. japonica (Hasegawa et al. 1983) and the North

American eel. A. rostrata (Khosla et al. 1985). Unlike tetrapods, teleost ANG I contains

the amino acid asparagine (Asn). instead of aspartate (Asp) in position one (Table 1). In

the North American eel. [~sn'-Arg~-~al~-Tyf-~al~-~is~-~ro~-Phe~-GIy' -Leu'O] ANG I

predominates (Khosla et al. 1985). However. a small proportion of eel ANG I contains

Asp in position 1. perhaps due to the enzymatic interconvenion of Asn to Asp (Hayashi

et al. 1978. Takemoto et al. 1983. Hasegawa et al. 1983a. Khosla et al. 1985). Position

nine appears to be variable in teleosts and it may be occupied by Asn. as in the chum

salrnon (Takemoto et al. 1983). histadine as in the goosefish (Hayashi et al. 1978). or

glycine as in the eel (Hasegawa et al. 1983. Khosla et al. 1985).

Table 1. Primêry structure of ANG I from select vertebrate spelleS

SDecies ino acid Sequence

Mammals Human, pig, horse, sheep rat, mouse, rabbit, guinea pig Ox

Asp Arg Val Asp Arg Val Asp

Tyr Ile His Pro Tyr Ile His Pro

Val

Phe His Leu Phe His Leu

His

Akagi et al., 1992 Fernley et al., 1986 Akagi et al., 1 992

Birds Fowl (Gallus domesticus ) Quail ( Coturnix coturnix japonica )

Val Val

Ser Ser

Nakayama et al., 1973 Takei and Hasegawa, 1990

Reptiles Snake (Elaphe climocophora ) Turtle (Pseudemys scripta ) Alligator (Alligator mississipiensis )

Val Val Val

Ser His Ala

Nakayarna et al., 1977 Hasegawa et al., 1984 Takei et al,, 1993a

Amphibians Bullfrog (Rana catesbeiana ) Hasegawa et al., 1983a Val Asn

Teleost Fishes Salmon (Oncorhynchus keta ) Eel (Anguilla jeponica ) Eel ( A. rostrata ) Goosefish (Lophius litulon )

Asn Asn Asn Asn

Val Val Val Val

Asn G ~ Y G ~ Y His

Takemoto et al., 1983 Hasegawa et al., 1983b Khosla et al., 1985 Hayashi et al., 1978

Elasmobranc h Fis hes Dogfish (Triekis syllia ) Asn Pro Takei et al,, 1993b Gln

Along with the well-established pressor effects of exogenous ANG I and ANG II,

investigators have further explored the mechanisms underlying the regulation of the

teleost RAS. Hemorrhage is a potent stimulator of plasma renin activity in the rainbow

trout, 0. Mykiss (Bailey & Randall 1981)- Acute hemorrhage has been found to

increase plasma ANG II levels in the Japanese eel. Anguilla japonica (Takei et al.

1988). Therefore a putative baroreceptor mechanism, one that senses decreases in

arterial blood pressure is thought to control the activation of the RAS in teleosts.

Environrnental changes that can lead to extracellular fluid loss also stimulates the

teleost RAS. In the tilapia, Tilapia mossambica, JG cells increase in size and number

following transfer to seawater (Krishnamurthy & Bem 1973). Plasma renin activity can

also increase in teleosts following seawater transfer (Henderson et al. 1 976, Sokabe et

al. 1973. Jackson et al. 1977. Arillo et al. 1981 ). In eels, transfer to seawater has been

found to increase plasma ANG Il levelsflakei et al. 1988).

In mammals. the extrarenal RAS is an area of active investigation (see review by

Johnson 1990). The synthesis of renin has been found to occur locally in many tissues

and organs, such as the brain. ovaries, blood vessels and the heart. The local

activation of the RAS and the consequent formation of ANG II rnay facilitate both

endocrine and paracrine functions. In teleosts. the CS and perhaps the ovaries have

been implicted as important sources of extrarenal-renin (Sokabe et al. 1970, Mandich 8

Massari 1994).

The corpuscles of Stannius (CS) of teleost fishes

(A) RAS and the CS

Since the discovery of the CS in 1839 by Stannius (see Pang 8 Schreibman 1986).

many theories have been proposed that have Iinked the CS to osmoregulation in

teleostean fishes. Rasquin (1956) observed that the activity and the size of the CS

increased in relation to the salinity of the water. Until the discovery of the interrenals,

the CS were thought to be the adrenocortical homologue of teleost fishes (De Smet

1962. Sandor et al. 1966. Youson et al. 1976). The CS were also thought to be the

parathyroid glands of teleost fishes. until this theory was also abandoned owing to a

lack of strong supporting evidence (Parsons et al. 1978, Shoumura et al. 1983. Lopez

et al. 1 984b, Milet et al- 1984). It has since been shown that the CS, at least in teleosts,

contain a powerful renin-like pressor substance (Chester-Jones. 1 966. Sokabe et al.

1970). The CS also contain stanniocalcin (Lafeber et al. 1988a.b. Wagner et al. 1988a.

1993). a 56 kDa glycoprotein (So & Fenwick 1977, 1979) that can suppress gill Mg2+ -

Ca2* activated ATPase (Ma & Copp 1978) to cause hypercalcemia.

In 1966. Chester-Jones was the first investigator to describe a link between the

RAS and the CS. in teleosts. Using the rat bioassay, he demonstrated that CS extracts

from freshwater eelç. A. anguilla, produced a powerful. angiotensin-like pressor

substance. He also showed that removal of the CS (stanniectomy) caused a rapid and

profound decline in the blood pressure of the stanniectomized eel (Chester-Jones et al.

1966). Four years later. these findings were confimed by studies on 3 additional teleost

species (Sokabe 1970). Histological cornpansons between the granulated cells of the

CS and JG cells of the kidney reveal numerous structural simitities (Sokabe & Ogawa

1974). Both ANG I and ANG II have been isolated from the CS of Japanese eels. A.

japonica. chu m salmon. 0. keta. Japanese goosefish. L. litulon. rainbow trout. 0.

mykiss (Ogawa 1982. Takemoto et al. 1983. Hasegawa et al. 1984b. Yamada 8

Kobayashi 1987).

Most recently. in the freshwater North American eel. A. rostrata. stanniectorny

led to a 45% reduction in donal aortic blood flow and a consequent 15% reduction-in

dorsal aortic blood pressure (Butler & Oudit 1995). Stanniectomy can also lead to

hypercalcemia. hypocalcemia. hypematremia. hyperkalemia and hypophosphaternia

(see reviews by Hirano 1989). In a rarely cited study. Ogawa (1968) showed that the

dramatic changes in plasma electrolyte levels following stanniectomy in goldfishes.

Carassius auratus. coufd be corrected simply with i.v. injections of ANG II. Thus. it has

been proposed that stanniectomy disrupts plasma electrolyte levels by impairing the

RAS. leading to alterations in normal blood flow to osmoregulatory organs in the

peripheral circulation. such as the gills. kidney and skin (Ogawa 1968. Butler et al.

1995, Butler 8 Cadinouche 1995).

(8) Distribution of the CS

The CS are small. ovoid endocrine glands unique to teleostean and holostean fishes.

Embryonically. the CS may anse from the pronephric, mesonephric or opisthonephric

ducts (Garrett 1942. Ford 1959. Belsare 1973a. Krishnarnurthy 1967). A nurnber of

important generalizations may be made with regard to the anatomical distribution of the

CS. In the more primitive holosteans. such as the bowfin and garpike. the CS tend to be

small. numerous and widely distributed throughout the kidney mass (Garett 1942.

Bauchot 1953. De Smet 1962. Youson & Butler 1976). In teleosts. such as the eel, the

CS tend to be larger. and occumng as a single pair embedded in the ventral surface of

the posterior mesonephric kidney (Bauchot 1953). There are notable exceptions to this

general trend. For instance. only one corpuscle is present in the ancient Notopterus

notopterus (Belsare 1973a). whereas in the relatively modem salmon. up to 14

corpuscles have been reported (Krishnamurthy 1976).

(C) Vascularizafion of the CS

The cells of the CS are arranged in strands and lobules that are separated by

connective tissue septa which also support the vascular and the nervous elements

(Krishnamurthy & Bern 1969). The CS is supplied by an extensive network of blood

vessels that can adapt to accommodate changing metabolic demands (Johnson 1972.

Wendelaar Bonga et al. 1977. Bhattacharya 8 Butler 1978). In the species examined

thus far. there appears to be considerable variation in the blood supply to the CS. In the

mullet. Mugli cephalus. branches from the renal arteries and the cardinal vein supply .

the glands (Johnson 1972). In the stickleback. Gasterosteus aculeatus, the CS receives

blood. rnainly from segmental veins of the dorsal musculature (Wendelaar Bonga et al.

1977). Regardless of the species. al1 the glandular cells of the CS recieve a rich

vascular supply. reflecting perhaps an important secretory function of the cells of the

CS.

(D) Innervation of the CS

The CS are richly innervated by both sympathetic and parasympathetic efferents, which

run parallel to the blood vessels supplying the glandular tissues (Heyl 1970, Wendelaar

Bonga et aï. 1977). However, there is no evidence of direct synaptic contact between

the nerve fibres and the cells of the CS (Heyl 1970. Krishnamurthy 8 Bern 1971,

Wendelaar Bonga et al. 1977, Bhattacharya & Butler 1978). lnstead, the nerves are

confined to the interlobular connective tissue septa. where they terminate on- adjacent

blood vessels and presumably modulate local blood flow. Fluorimetric techniques show

that these fibres can be either cholinergic or adrenergic; carrying the neurotransmitten

acetylcholine, noradrenaline or serotonin (Unsicker et al. 1977).

(E) Cell Types of the CS

The CS of teleostean fishes contain 2 distinct secretory ce11 types, the type-1 cells and

type-2 cells (Wendelaar-Bonga 8 Greven 1975, Wendelaar-Bonga et al. 1977). An

additionai neurosecretory cell type has been identified in the CS of the white sucker,

Catastomus commersoni (Marra et al. 1 998). The structure of type-1 cells resemble that

of other cells active in the synthesis and secretion of polypeptides, such as cells of the

exocrine pancreas. The type-1 cell is ovoid, contain large nucleus with a pronounced

nucleolus, extensive granular endoplasmic reticulum, large Golgi bodies and

characteristic large, round, densely-staining secretory granules. In al1 species

examined, the majority of CS cells are of the type-1 cytology (Oguri 1966. Fujita 8

Honma 1967, Carpenter & Heyl 1974. Cohen et al. 1975). In the bowfin, toadfish and a

number of other species (Youson & Butler 1978. Wendelaar Bonga 8 Greven 1975.

Bhattacharya 8 Butler 1978). the CS is composed alrnost exclusively of type4 cells.

The other cell type. type2 cells. tend to be narrower. with smaller. itregularly

shaped granules. sparsely distnbuted endoplasmic reticulum and fewer golgi bodies

(Wendelaar Bonga 8 Greven 1975, Wendelaar Bonga et al. 1977). There is evidence.

that the two cell types corne from a single lineage. whereby their histologicals

differences may reflect different stages of development or different States of activity

(Kaneko et al. 1992. Bhattacharya & Butler 1978, Youson & Butler 1976, Bhattacharya

et al. 1978). It has even been suggested that type-2 cells rnay represent type-1 cells

that are undergoing programmed cell death. or apotosis (Wyllie et al. 1980).

Pharmacological inhibition of the RAS

The RAS plays a major role in the regulation of blood pressure. as well as fluid and

electrolyte homeostasis. It is a closed-loop. negative-feedback system responding to

volume or sodium depletion and to factors modifying renal blood fiow. Two key

enzymes in the RAS cascade are the highly specific aspartyl protease. renin. and by

contrast, the highly non-specific ACE, a dipeptidyl-carboxypeptidase. Renin hydrolyses

the globular plasma protein. angiotensinogen. to release the decapeptide. ANG 1. ANG

I is subsequently converted to the powerful vasoconstrictive octapeptide. ANG II by

ACE. ANG II is hydrolyzed further by arninopeptidases to become ANG III. and other

relatively inactive peptides.

In theory, pharmacological interference with the RAS is possible at every step in

the formation or action of ANG II. However. at present. drugs have been developed to

block the RAS at only 3 steps. Firstly. renin inhibitors selectively block the formation of

ANG I from renin-substrate. Secondly, AC€ inhibitors prevent the formation of ANG il

from ANG 1. Thirdly. ANG II receptor antagonists inhibit the cellular responses to ANG

II. ANG II analog receptor-antagonists such as [Sar'. Ilel-ANG II (Sarile) have helped to

clarify the role of the RAS in normal as well as pathological models. More recently.

nonpeptide ANG II antagonists such as Losartan. have gained rapid and widespread

acceptance clinically and experimentally. interestingly, the biosynthesis, secretion and

maturation of renin are potential sites of inhibition that have not yet been explored.

Given the intense medical interest in the RAS, much of the research on the

biochemical properties of the RAS and its interaction with chemical inhibitors has

focused primarily on rnammalian models. To date. the amino acid sequence of renin

has only been determined for the rat, mouse and human (Hirose & Murakami 1992). In

recent years. tremendous strides have been made in our understanding of the

biologicat and the biochemical properties of the RAS in mammals. However, the role of

the RAS and the effect of RAS inhibitors on the cardiovascular regulation of teleost

fishes have not been examined in depth.

(A) Inhibition of renin

Renin belongs to the aspartyl protease family of enzymes that incfudeç pepsin.

prochymosin and penicillopepsin. Renin is distinct from other aspartyl protease due to

its high specificity for its substrate, angiotensinogen. Renin occurs naturally in both

active and inactive forms (Lumbers 1971). In the hog kidney. 3 foms of renin have

been isolated (Inagami 8 Murakami 1977): a 42-kDa active renin. a 61-kDa prorenin

and a 140-kDa inactive renin. In hurnans, it is estimated that between 50 to 60 % of

circulating renin is in the f o m of prorenin or inactive renin (Boyd 1977). Renin-binding

protein, located in the renal tubules of rats. has been implicated in the interconversion

of active and inactive renin (Ikemoto et al. 1982).

Angiotensinogen. or renin substrate. is a 58 kDa hepatic glycoprotein that has

been isolated in humans, as well as a handful of other mammals (Schiffrin 8 Genest

1983). In mammals, the ANG I decapeptide is liberated from the N-terminal of

angiotensinogen by renin. through the hydrolytic cleavage of a leucine-valine bond

called the "scissile" bond (Tewsbury et al. 1981). As is the case with renin, there also

appears to be variable forms of angiotensinogen (Murakai et al. 1984). In humans.

variations in the primary structure of angiotensinogen are thought to be the result of

different pathways of post-translational processing and of noncovalent interactions

between the newly secreted angiotensinogen molecule and other circulating moieties

(Campbell et al. 1985). Two enzymes. a cardiac chyme and tonin. an enzyme found in

the mouse subrnaxillary gland, can synthesize ANG II directly from renin substrate,

without the participation of AC€ (Erdos 1976, Urata et ai. 1990).

Prior to the development of ACE inhibitors and nonpeptide ANG II receptor

antagonists, numerous attempts have been made to synthesis renin inhibitors for

therapeutic applications- Five different categories of renin inhibitors are descnbed in the

literature: phospholipid inhibitors, renin antibody inhibiton, angiotensinogen-analogue

inhibitors. prorenin inhibitors and the most wideiy studied renin inhibitor. Pepstatin A.

Membrane-derived phospholipid preinhibitors. such as PE-140, were the first renin

inhibitors to receive serious consideration (Sen et al. 1969a. b). PE-140 blocked the

pressor effects of exogenous renin and lowers blood pressure in dogs (Antonaccio

1982. Davis et al. 1974). Specific antibodies to renin were also considered as

candidates for renin in hibitors. l nitial findings showed that the antibodies can in hibit

renin activity both in vitro and in vivo (Ondetti et al. 1982. Kohler et al. 1975).

Chemically modified angiotensinogens have also shown promise as renin inhibitors.

Modifications are made to the "scissile" bond. thereby preventing renin from carrying

out the proteolytic release of ANG I (Burton et al. 1975. Poulsen et al. 1973).

Alternatively. fragments of prorenin from the mouse submaxillary-gland have also been

found to have inhibitory effects on renin (Panthier et al. 1982). Unfortunately. none of

these four strategies have proven to be effective in the long tem.

In 1971. a bacterially derived peptide. Pepstatin A. was introduced as a

nonspecific inhibitor of acid proteases. such as renin (Marciniszyn et al. 1976, Aoyagi et

al. 1971 ). Of the five different classes of renin inhibitors. Pepstatin A is the most

effective. Pepstatin A blocks the pressor effects of exogenous renin and renin

substrate. without interfering with the actions of ANG I or ANG II. The other major

physiological effects of renin. attributable to the de novo synthesis of ANG I and ANG II.

can also be blocked by pepstatin A. Currently. Pepstatin A is one of the only renin

inhibitors that are commercially available. The effect of Pepstatin A on the RAS has not

been exarnined in fishes.

(B) Inhibition of ACE

ACE is a large. 150 to 206-kDa peptidyl carboxypeptidase that is found in the lung,

kidney and plasma of mammals (Peach 1977). Unlike renin, ACE has a wide substrate

specificity (Ehlers et al. 1989, Erdos & Skidgel 1987). enabling it to hydrolyze ANG I .

bradykinin, enkephalins, neurotensin, substance P as well as LHRH (see review by

Erdos 1976). Recently. 2 forms of ACE have been identified. One occurs in the

endothelium of the lung and kidney, and the other in germinal cells of the testes (Ehler

& Riordan 1990, Beldent et al. 1993. Hooper 1990 ). In the RAS, ACE is responsible for

the final proteolytic step in the formation of ANG II. ACE cleaves the two C-terminal

amino acids from ANG 1. to form the ANG II octapeptide.

Although ANG 1 does not stimulate vascular smooth muscle contraction, it can

act directly on the central nervous systern, adrenal cortex, adrenal rnedulla and the

kidney (8uckley 1972. Swanson et al. 1973, Saruta et al. 1972. ltskowitz 8 McGriff

1974). The discovery of ANG 1-specific binding sites support these findings (Goodfriend

et al. 1972). ANG l has a highly conserved structure. Between species, variations in its

primary structure is restricted almost exclusively to positions 1, 5 and 9 (Table 1).

Among mammals, the ANG I of ox is unique because it contains valine, instead of

isoleucine in the fifth position (Akagi et al. 1982). This amino acid configuration of ANG .

I is characteristic of submammalians. not of rnammalians.

ACE inhibitors such as captopril. mask the catalytic properties of AC€ by

competitively binding to its active site (Ehlers 8 Riordan 1989, 1991). In mammals,

ACE-inhibition decreased plasma ANG II and aldosterone levels (Johnson et al. 1979,

Hulthen 1978). causing total peripheral resistance and arterial blood pressure to decline

(Lund-Johansen et al. 1984). Similar findings have also been reported in

nonmammalian species. Butler et al. (1995) showed that in the bowfin. A. calva.

captopril can attenuate the pressor response to exogenous ANG 1.

(C) Inhibition of ANG II Receptors

ANG II has been implicated in the pathogenesis of vanous cardiovascular disorders that

affect millions of people(Peach 1977). Synthetic ANG II analogs, which act as

competitive antagonists of ANG II receptors, have proven to be clinically and

experimentally relevant (Khosla et al. 1974). Such cornpounds. including [Sar'. Ileu]- -

ANG II (Sarile) and [Sar'. Ala7-ANG II (Saralasin) inhibit the acti-ons of ANG II at the

receptor ievel (Brown et al. 1983, Timmermans et a/. 1992) and has been shown to

bind al1 ANG II receptor subtypes. ubiquitously (Khosla et al. 1974). Hence, these

compounds are ideally suited for inhibiting the biological actions of ANG II and for

identifying its specific sites of action.

Tne recent development of specific. nonpeptide ANG II receptor antagonists, in

particular Losartan. PD1 2331 9, PD1 231 77 and CGP42112A have enabled researchers

to characterize different subtypes of ANG II receptors on the basis of their affinity for

these compounds. Losartan (Dup 753) binds the AT, receptor subtype (Timmermans et

al. 1993). while CGP42112A. PD1 231 77 and PD1 2331 9 bind preferentially to receptors

of the AT- subtype (Chiu et al. 1989. Burnpus et al. 1991 ). Since AT, recepton mediate

al1 the known actions of ANG II. their inhibition by Losartan can block al1 the effects of

ANG II on vascular srnooth muscle, the kidney. and the adrenal cortex (Timmermans et

al. 1993). Although studies have been undertaken to identify AT, receptors in

nonmammaiian species, the physiological role of AT, receptors in nonmammalians has

yet to be established. The role of AT, receptors, in al1 species, is unclear (Saavedra &

Timmermans 1994). In mammals, the use of AT, receptor antagonists such as

PD1 231 77. PD12331 9 and CGP42112A do not affect any of the actions of ANG II (Chiu

et al. 1989, Whitebread et al. 1989). A study on the effects of AT, and AT, inhibition, in

a piscine species, has not been previously undertaken.

The purpose of this investigation is to test the hypothsis that the CS. in the North

American eei (Anguilla rostrata Lesueur), is an important source of renin-like enzyme

activity. The effect of i.v. injections of CS extract (CS-EXT) on blood flow in the dorsal

aorta and the caudal vein will be explored. Furthermore. phannacological antagonists of

the RAS will be tested in the eel, to determine their effects on the dorsal aortic and

caudal venous blood flow responses to CS-EXT. In addition. this is the first study to test

the effects of subtype specific. nonpeptide antagonists of ANG II receptors. in fishes.

MATERIALS AND METHOD

Experimental animals

Female North American eels. (Anguiiia rostata LeSueur), weighing 800-1200 g were

collected in October 1996, from the St. Lawrence River near Quebec City by Pecheries

Gingras. DuPont. St. Nicholas. Quebec. Canada. and shipped by air to Toronto.

Ontario. They were delivered to the Department of Zoology. animal care facilities.

where they were held in aerated plastic holding tanks (25 fish per 5001 capacity tank),

supplied with aerated. dechlorinated, Toronto tap water (Na', 0.45; CI- 0.95; K' 0.02;

Ca2-. 0.98; Mg2'. 1 -59 mmol 1") at 1 1 .O + 1.0 O C . Eels were held under a 12 : 12 h

light-dark cycle and fed woms ad libiurn. Eels were selected at random for expenments

that started in March 1997.

Experimental set-up and surgical procedures

Eels were selected at random and placed individually in Plexiglas observation

chambers (13.8 x 12.0 x 114.5 cm ) supplied with aerated, dechlorinated tap water

(12.0 + 0.5' C). Eels were adapted to the observation chambers for at least 3 days

before the experiment started.

(A) Insertion of Doppler flow probes

Eels were anaesthesized in a solution of methane tricanesulphonate (MS222) in tap

water (1 g 1'' ). When movement of the operculum ceased, the eel was deemed to be

unconscious. and it was then wrapped carefully in a wet Cotton towel and placed on its

back on an enamel surgical tray. A ventro-medial incision into the coelomic cavity was

started near the anterior border of the liver, and extended 5-6 cm posteriorly. The

edges of the body wall were retracted and the liver was deflected to the right to expose

the dorsal aorta. Next. the dorsal aorta was carefully dissected free of connective

tissue. care being taken not to break the fine intersegmental arten'es supplying the body

wall. The dorsal aorta varied in diameter according to the size of the eel. so in each

case. it was fitted with a probe beanng a cuff diameter of 2.6. 2.8. or 3.0 mm. Signal

transmission was improved if ultrasonic transmission gel (Aquasonic 100. Parker Labs..

New Jersey) was smeared on the contact surface between the transducer crystal of the

probe and the wall of the artery. Pieces of sterile Gelfoam measuring 3 mm X 5 mm

were packed against the outside of the cuffed vesse1 to provide support and to reduce

any bleeding. The lead coming frorn the flow probe was loosely stitched to the body

wall to hold it in the correct position. Then the cut edges of the body wall were pulled

together with size 3-0 Chinese braided silk sutures. Finaliy. the skin was sutured with

size 3-0 stainless steel wire.

The caudal vein was also fitted with an appmpriately sized Doppler probe.

Access to the caudal vein was made through an 5-cm ventro-medial incision through

the body wall along the anterior border of the caudal fin. The edges of the body wall

were retracted to expose the caudal vein. The vein was stripped free of connective

tissue; great care being taken to not tear the segmental veins draining into it.

Application of gel was followed by insertion of a suitable sized Doppler cuff which was

tied in place in much the same manner as the dorsal aortic cuff. The surgical area was

packed with small. 3 x 5 cm pieces of Gelfoam sponge. Then the cut edges of the body

wall were pulled together with size 3-0 Chinese silk sutures. Finally. the skin was

sutured with size 3-0 stainless steel wire.

When the surgery was completed, the eel was nnsed with tap water to remove

any residual anaesthetic. It was then given a 2-3 ml injection of Ampicillin (100 mg. ml"

i.m.). When the eel was able to swim again. it was transferred to an observation

chamber that was covered with a black plastic sheet. 1 ml of Ampicillin (100 mg. ml"

i.v.) was deliver i.v. daily via the caudal vein catheter for the duration of the

experimental period. Each eel was allowed to rest for 3-4 days before experiments

commenced. to allow its cardiovascular parameters to return to normal. There was no

evidence of infection during the next four days and the wound healed well with no

apparent leakage or discomfort for the eel.

(B) Collecting the corpuscles of Stannius and posterior kidney tissue

Each eel was randomly selected from stock. anaesthetized with MS 222 (1 .Og. 1-') and

placed on a wet cotton towel. It was rolled over onto its side and a 5 cm incision

through the body wall was made a few mm below the Iateral Iine and parallel to it,

immediately above the ventral fin. The cut edges were retracted to expose the edge of

the posterior portion of the kidney. The bladder was iifted away from the ventral surface

of the kidney by btunt dissection, with no evident bleeding. The oval, ivory-coloured CS,

measuring approximately 2-3 mm in diameter and weighing approximately 6 mg. could

now be seen as embedded in the surface of the kidney, at the point where the right

posterior cardinal vein emerges from the kidney surface. Both corpuscles were easily

excised and then placed on ice. where they were immediately transferred to a -50 O C

refrigeration compartment. The bladder was carefully fowered back into place before

the edges of the body wall muscle were sutured together with size 3-0 Chinese braided

silk. Following this. the skin was sutured with 3-0 stainless steel wire. Finally the eel

was given a 2-3 ml injection of Ampicillin (100 mg.ml-' im.) and retumed to a 400 liter

glass aquarium supplied with constantly flowing aerated dechlorinated tap water (12.5 2

0.5 OC). until it was used for additional blood flow experiments.

Sarnples of posterior kidney tissue adjacent to the corpuscles were removed

from five eels. The tissue was frozen immediately. later to be thawed. weighed and

extracted on the day of the experiment. The posterior kidney extracts were prepared

using the same method used to prepare extracts of the CS. Afterward, the eel was

killed with an overdose of MS 222.

(C) Insertion of drug delivery catheter

A 3 cm incision was made 3 mm below and parallel to the lateral line near the tip of the

caudal fin. Muscle tissue was gently plied apart and retracted to expose a 2 cm length -

of caudal vein. It was freed of connective tissue and bone of the neurohaemal arch. A

heparinized polyethylene catheter (PE-10. Clay Adams) was inserted into the caudal

vein and pushed 10-12 cm anteriorly. The catheter was then tied in place with 5-0

braided silk sutures before the incision was closed with 5-0 braided silk sutures. This

left a trailing length of approximately 20 cm. The end of the catheter was heat-sealed.

The catheter was used later for the delivery of al1 the dwgs and hormones without

disturbing the eel during the experiments. It was flushed regularly with 0.4 ml heparin-

solution to prevent the formation of blood clots.

(D) Calibration of Doppler flow probes

A postmortem calibration of dorsal aortic and caudal venous flow probes was performed

in situ. This allowed the experirnenter to calculate the absolute values for blood fiows

(Butler & Oudif 1995, Oudit & Butler 1995). At the end of the flow experiment. the eel

was killed using a 5 ml injection of Somnotol (sodium pentobarbital. 65 mg.ml-' i.v.). The -

ventral incision was re-opened to gain access to the Doppler fiow probe on the dorsal

aorta. First. the dorsal aorta was exposed and catheterïzed 5-10 mm anterior to the

probe with PE-100 (i. d. 0.86 mm, o. d. 1.5 mm) polyethylene tubing for blood perfusion.

It was placed close to the probe so as to minimize leakage through the segmental, as

well other arteries. Then the catheter was tied in place with 3-0 silk sutures. An oufflow

catheter, also PE-100, was inserted into the dorsal aorta posterior to the probe and

positioned 5 mm behind it. Then the catheter was tied in place with 3-0 silk sutures. The

Doppler flow probe on the caudal vein was prepared for calibration in a similar manner.

There was no sign of hemorrhaging in the region of the probe. nor of coagulated blood

or other obstructive tissues on the contact surface between the probe cuff and the

blood vessels. Connective tissue growth surrounding the Doppler probes provided

additional stability for the duration of the experiments and the calibrations.

Freshly collected, heparinized eel blood was infused using an infusion pump

(Harvard Apparatus Infusion Pump). over a range of flow rates that encompassed the

minimum and the maximum values for each eel (Butler 8 Oudit 1995a. b). Flow rates

were analyzed by linear regression analysis to provide calibration curves for the dorsal

aortic probe and the caudal venous probe. These plots confirmed that proper acoustic

coupling was maintained during the calibration procedure.

Drugs and peptides

€el Angiotensin I = [Asn'. Val5. Glyq-ANG I (MW=1200.7); Eel Angiotensin Il = [Asn'.

Val5]-ANG II (MW=lO30.5); €el Angiotensin I I I = pal4}-ANG I I I (MW=W 6.5); Human

renin substrate tetradecapeptide = [l-141-hRS (MW=1758.9), Pepstatin A (MW= 685.9)

and Sarile = [Sar'. Ile7-ANG II (MW=967.6) were al1 supplied by Penninsula

Laboratories (Belmont. CA). Captopril (SQ 14 225, MW = 217.28) was supplied by

Bristol-Myers Squibb Pharmaceutical Research lnstitute (Princeton, NJ). The ANG II

receptor blockers were supplied as follows: Losartan potassium (DuP 753. MK 954

MW=461). Dupont Merck Research and Development. Wilmington, DE.; PD123319

(MW=749.4). Parke-Davis Pharmaceutical Research Division. Ann Arbor, MI. Methane

tricainesulfonate (MS 222). Sigma Chemical Corp.. St. Louis. MO.; Somnotol (Sodium

pentobarbital in isotonic saline). MTC Pharrnaceuticals. Cambridge. Ont.. Canada:

Ampicillin sodium. Novopharm. Toronto. Ont-. Canada; Heparin solution. Hepalean-

Organon Tekita. Toronto. Ont.. Canada. All of the drug and peptide injections were

made with 0.9% NaCl-

Blood flow experiments: Experimenfal protocol

Series /: Response to components of the RAS

Experiment 1: [ A d , Val5, GlyT-ANG 1. 5. 10. 20. and 5Gng. kg bw-' doses of [Asn'.

Val5. GIS]-ANG 1 were injected i.v. in 0.2 ml of 0.9% NaCl to test the effect on donal

aortic and caudal venous blood flows (n=5). These test doses were not injected until

flow rates had stabilized and remained at baseline levels for 45 minutes. The caudal

venous dnrg delivery catheter was flushed with 0.2 ml of 0.9% NaCl after each peptide

injection to ensure that al1 the peptide reached the bioodstream. When blood flow had

returned to normal. after the effect of the peptide had subsided, the animals were

injected with a second 0.50 ml of 0.9% NaCl solution to show that volume loads had not

affected blood flows. This basic experimental protocol was used also for experiments 2,

3. 4 and 5 which are described in this section. Following each expenment, the animals

were given a one-day recovery period.

Experiment 2: [ A d , Valq-ANG II This expenrnent measured the effect of 5, 10, 20,

and 50 ng kg bw-' doses of [Asn'. val)-ANG II. in 0.2 ml of 0.9% NaCI. on dorsal aortic

and caudal venous blood flows (n=5).

Experiment 3: Human Renin Substrate (hRS) This experiment measured the effect

of 50, 100, 150 ng kg bw-' hRS. in 0.2 ml of 0.9% NaCI, on dorsal aortic and caudal

venous blood flows (n=5).

Experiment 4: pal4]-ANG III This experiment measured the effect of 5. 10. 20. 50 ng

kg bw" [Val4]-ANG III. in 0.2 ml of 0.9% NaCI. on dorsal aortic and caudal venous blood

flows (n=5).

Experiment 5: Eel corpuscle of Stannius extract (CS-EXT) This experiment

rneasured the effect of extracts of 0.50, 1.25, 2.50 mg of eel corpuscles of Stannius

(CS-EXT) .kg bw-'. delivered in 0.2 ml of 0.9% NaCI, on dorsal aortic and caudal

venous blood flows (n=5).

Experiment 6: Eel posterior kidney extract (PK-€Xi) This experiment measured the

effect of an extract of 5 mg of eel posterior kidney (PK-EXT).kg bw", delivered in 0.2 ml

of 0.9% NaCI. on dorsal aortic and caudal venous blood flows (n=5).

Series II: Response to RAS antagonists

Experirnent 1: Effect of Pepstatin A on the blood flow response to [ Asn'. Vals.

Gly9]ANG I This experiment measured the effect of 1 mg kg bw-' dose of Pepstatin A.

delivered i-v. in 0.5mI of 0.9% NaCl, on the dorsal aortic and caudal venous blood flow

response to 50 ng kg bw*' dose of ANG I i.v. Dorsal aortic and caudal venous blood

Rows were measured continuously. The drug delivery catheter was flushed with 0.2 ml

of 0.9% NaCl after each injection to ensure that al1 the peptide or antagonist reached

the bloodstream. Eels were injected with 0.50 ml saline solution (0.9% NaCI) to

measure the effect of volume loads on blood flow. 55 minutes later, eels were injected

with 50 ng kg bw" dose of [ Asn'. vals. Glfl-ANG 1. After a further 45 minutes. when

the flow rates had retumed to preinjection levels. the eels were given a 1 mg kg bw-'

dose of Pepstatin A. Eel was injected with 50 ng kg bw-' dose of [ Asnl. Val5. Glfl-

ANG I 10 minutes later. Flow rates were measured for an additional 40 minutes, until

rates had returned to preinjection levels- The basic expenmental protocol for this

experiment was also used for experiments 2. 3.4. 5 .6 .7 . 8.9. 10. 11. 12, 13 and 14 on

this section (n=5).

Experiment 2: Effect of Pepstatin A on the blood flow response to [ Asn', Val79

ANG II This expenment measured the effect of a 1 mg kg bw-' dose of Pepstatin A.

delivered i.v. in O.5ml of 0.9% NaCI, on the dorsal aortic and caudal venous blood flow

response to a 50 ng kg bw-1 dose of [ Asnl. ValYANG II (n=5).

Experiment 3: Effect of Pepstatin A on the blood flow response to human renin

substrate (hRS) This experiment measured the effect a of 1 mg kg bw-' dose of

Pepstatin A. delivered i-v. in 0.5ml of 0.9% NaCl, on the dorsal aortic and caudal

venous blood flow response to a 150 ng kg bw-' dose of hRS (n=5).

Experirnent 4: Effect of Pepstatin A on the blood flow response to extract of

corpuscles of Stannius (CS-EXT) This experiment measured the effect of a 1 mg kg

bw-' dose of Pepstatin A. delivered i-v. in 0-5ml of 0.9% NaCI, on the dorsal aortic and

caudal venous blood flow response to a 2.50 mg kg bw-' of CS-EXT(nr5).

Experiment 5: Effect o f Captopril on the blood flow response to [ A d , Vals, Glfl-

ANG I This experiment measured the effect of a 1 mg . kg bw-' dose of Captopril.

delivered i.v. in 0.5ml of 0.9% NaCI, on the dorsal aortic and caudal venous blood flow

response to a 50 ng kg bw-' dose of [Asnl. Vals. GIfl-ANG I (n=5).

Experiment 6: Effect o f Captopril on the blood flow response to ( Asnl, ValSI-ANG

Il This experirnent measured the effect of a 1 mg kg bw-' dose of Captopril. delivered

i-v. in 0.5ml of 0.9% NaCI. on the dorsal aortic and caudal venous blood flow response

to a 50 ng kg bw-' dose of [Asnl. Val5]-ANG ll(n=5) .

Experiment 7: Effect of Captopril on the blood flow response to hurnan renin

substrate (hRS) This experiment measured the effect of a 1 mg kg bw-' dose of

Captopril. delivered i.v. in 0.5mI of 0.9% NaCI, on the dorsal aortic and caudal venous

blood flow response to 150 ng kg bw" dose of hRS (n=5).

Experirnent 8: Effect of Captopril on the blood flow response to extract of

corpuscles of Stannius (CS-EXT) This experiment measured the effect of 1 mg kg

bw-' dose of Captopril. delivered I.V. in 0.5ml of 0.9% NaCI. on the dorsal aortic and

caudal venous blood flow response to 2.50 mg kg bw-' dose of CS-EXT (n=5).

Experiment 9: Effect of (Sar1, Ile8]-ANG II (Sarile) on the blood flow response to

[Asn', Val5]-ANG II This expenment measured the effect of a 50 pg . kg bw-' dose of

Sarile, delivered i-v. in 0.5ml of 0.9% NaCI. on the dorsal aortic and caudal venous

blood flow response to a 50 ng kg bw-' dose of [Asn', Val)-ANG II (n=5).

Experirnent 10: Effect of (Sar1, Ile8JANG II (Sarile) on the blood flow response to

extract of the corpuscles of Stannius (CS-En) This experiment measured the

effect of a 50 pg . kg bw-' dose of Sarile. delivered i.v. in 0.5ml of 0.9% NaCI. on the .

dorsal aortic and caudal venous blood flow response to a 2.50 mg - kg bw-' dose of CS-

EXT (n=5).

Experirnent 11 : Effect of Losartan on the blood fiow response to [ Asnl, ValSI-ANG

II This experiment measured the effect of a 5 mg kg bw" dose of Losartan. delivered

i.v. in 0.5mI of 0.9% NaCI, on the dorsal aortic and caudal venous blood flow response

to a 50 ng kg bw*' dose of [ A d . V~I~J-ANG II (n=5).

Experiment 12: Effect of Losartan on the blood flow response to extract of

corpuscles of Stannius (CS-EXT) This experiment rneasured the effect of a 5 mg kg

bw" bw dose of Losartan. delivered i.v. in 0.5ml of 0.9% NaCI, on the dorsal aortic and

caudal venous blood flow response to a 2.50 mg kg bw-' dose of CS-EXT(n=5) .

Experiment 13: Effect of PD123319 on the blood flow response to [ Asnl, ValSI-

ANG II This experiment measured the effect of a 5 mg kg bw-' dose of PD123319.

delivered i.v. in 0.5ml of 0.9% NaCI, on the dorsal aortic and caudal venous blood flow

response to a 50 ng kg bw-' dose of [ Asn', Val5]-ANG II (n-5).

Experiment 14: Effect of PD123319 on the blood flow response to extract of

corpuscles of Stannius (CS-EXT) This experirnent measured the effect of a 5 mg kg

bw-' dose of PD123319, delivered i.v. in 0.5rnl of 0.9% NaCI, on the dorsal aortic and

caudal venous blood flow response to a 2.50 mg kg-bw" dose of CS-EXT ( ~ 5 ) .

RESULTS

Calibra tion cuwes and velocity profiles

Dorsal aortic and caudal venous calibration curves for three freshwater eels are shown

in Figure 1. 80th curves have similar slopes and there was a strong positive linear

correlation between the Doppler shift (KHz) and the blood perfusion rate in ml.min-'.kg

bw-'. Dorsal aortic and caudal venous blood fiows were then obtained by interpolation.

Velocity profiles across the dorsal aorta and caudal vein (2.8 mm probe) of freshwater

eels were obtained by making small increments in the range outputs which determined

the distance at which the blood velocity was measured (0-10 mm) (Butler & Oudit

1995). During the present experiments, the range was adjusted accordingly so as to

measure the peak mean blood flow in both the dorsal aorta and caudal vein.

(A) ANGI,ANGIIorANGIlI and bloodflow

This experiment compared the blood flow responses (BFR) in freshwater eels to a

range of doses of each of three angiotensins including (Asn',ValS.Glg]-ANG I (ANG 1).

[Asn'.ValS]-ANG II (ANG-II). and val4]-ANG III (ANG III). The repeated measures

ANOVA showed that whereas ANG 1 and ANG II groups both produced significant

increases in caudal venous blood flow (CVBF) and dorsal aortic blood flow (DABF).

while the ANG III group had a more modest effect when compared with the saline

injected control group (Figure 2).

CVBF increased significantly following the i.v. injection of only 5 ng-kg bw-' of

ANG I or ANG II (Figure 2) but it was not until the dose reached 10 ng.kg bw-' of each

Figure 1. Caudal venous ( O ) and dorsal aortic ( ) calibration curves for

freshwater North Amencan eels, A. rostrata ( n = 3 ). Linear regression lines:

Y = Doppler shift ( Khz ), X = Blood flow rate (ml.rnirfl.kg bw-');

CV: Y = 0.08X+ 0.11, rr0.98. p <0.0001.

DA: Y =O.O8X +0.0004, r=0.99. p <0.0001.

Figure 2. CVBF ( 0 ) and DABF ( I ). in freshwater North American eels, A.

Rostrata, in response to i.v. injection of 5, 10.20 or 50 ng. kg bw-'of [ ~ s n ' , val5,

GI~~]-ANG 1, [Asn', ~al7-ANG II and [V~I"]-ANG III. CVBF mponse to ANG I

or ANG II: O P c 0.05 compared with saline control, 5. 10, 20 ng. kg bw-'

peptide; O P c 0.05 compared with saline control, 5. 10 ng. kg bw" peptide; O P

< 0.05 compared with saline control and 5 ng. kg bw-'. A P < 0.05 compared with

saline control. DABF response to ANG I or ANG II: P < 0.05 compared with

saline control. 5. 10. 20 ng. kg bw" peptide; . P < 0.05 compared with saline

control. 5 and 10 ng. kg bw-' (for ANG I and ANG II). A P c 0.05 compared with

saline control and 5 ng. kg bw". CVBF response to ANG III: O P c 0.05 .

compared with saline control. S. 10. 20 ng. kg bw-' peptide. A P < 0.05 compared

with saline control. 5. 10, ng. kg bw*' peptide. DABF response to ANG III: A P <

0.05 compared with saline control. 5. 10. 20 ng. kg bw-' peptide; ANOVA,

Duncan's Multiple Range Test. Values are mean + SEM; n = 5.

peptide that there was a significant increase in DABF. ANG III stood alone insofar as it

required 20 ng .kg bw-' of the peptide to increase significantly CVBF and 50 ng.kg bw"

to increase DABF (Figure 2). In both the ANG I and ANG II groups there was a dose

dependent increase in both CVBF and DABF.

(B) Tissue exfracts and blood flow

Posterior kidney extracts (PU-EXT), human renin substrate or human angiotensinogen

1-1 4 (hRS) and extracts of corpuscles of Stannius (CS-EXT) were each tested for their

effect on CVBF and DABF in freshwater eels. An i.v. injection of 5 mg of PK-EXT had

no measurable effect on either CVBF or DABF compared with saline injected controls

(Figure 3). An i.v. injection of 50 ng-kg bw" of hRS had no rneasurabie effect on CVBF

or DABF whereas 100 ng.kg bw" of the peptide was followed by a significant increase

in CVBF but not DABF. At the highest dose (150 ng-kg bw-') both CVBF and DABF

increased significantly compared with the saline injected controls (Figure 3).

Extracts of CS brought about important changes in fiows. An i-v. injection of

extract from 1.25 mg of tissue increased the CVBF from 2.80 + 0.18 ml. min".kg bw-' to

6.38 + 0.31 ml.min.kg bw-' (P ~0.05) and the DABF from 6.99 + 0.47 ml.min.kg bw" to

10.14 + 0.36 ml.rnin.kg bw-' (P ~0.05). The highest dose of extract of 2.5 mg of tissue

i.v. increased further both the CVBF and DABF to levels which were significantly

greater than the flows observed in both the saline injected controls and the 1.25 mg. kg

bw-' CS-EXT group.

Figure 3. CVBF ( ) and DABF ( ), in freshwater North American eels, A.

rostrata, in response to i-v. injection of 50, 100 or 150 ng. kg bw" of human

renin-substrate (hRS) and 0.5, 1.25 and 2.5 mg. kg bw-' extract of CS (CS-EXT)

expressed in units. kg bw-'.CVBF response to hRS: A P < 0.05 compared with

saline control, posterior kidney extract injection (PK-EXT) and 50 ng. kg bw-'

hRS. DABF response to hRS: A P c 0.05 compared with saline control, PK-

EXT, 50 and 100 ng. kg bw-' hRS. CVBF response to CS-EXT: Ci P < 0.05

compared with saline control, PK-EXT, 0.5 and 1.25 mg. kg bw-' extract; A P <

0.05 compared with saline control, PK-EXT, 0.5 mg. kg bw-' extract. DABF

response to CS-EXT: . P < 0.05 compared with saline control, PK-EXT, 0.5

and 1.25 mg. kg bw-' extract; A P < 0.05 compared with saline control, PK-EXT,

0.5 mg. kg bw" extract; ANOVA, Duncan's Multiple Range Test. Values are

mean + SEM; n = 5.

0.5 ml 5,O saline PK-EX1 (mg) CS-EXT (mg)

4- -

(C) Pepstatin A and the blood flow responses to ANG 1, ANG II. hRS or CS-

EXT

Figure 4 shows that CVBF and DABF both increased significantly in freshwater eels

following an i.v. injection of 50 ng-kg bw-' of ANG 1. Peak blood flow rate (BFR) in the

CV and DA were 10.32 t 0.23 and 12.44 t 0.49 rnl.kg bw-' respectively. The onset of

peak CVBF and DABF occurred 8 and 9 minutes after the injection of ANG I and

remained elevated for 28 and 18 minutes before retuming to the preinjection rates.

These patterns in flow were not affected measurably by the prior i.v. injection of 1

mg-kg bw-' of Pepstatin A (Figure 4). The Pepstatin A experiment was repeated using

50 ng.kg bw-' of ANG II (Figure 5). Again. there was a dear and significant increase in

both CVB and DABF both before and after an i.v. injection of 1 mg-kg bw-' of Pepstatin

A. These results were not unexpected since Pepstatin A is an effective renin inhibitor in

marnmals.

Next. 150 ng.kg bw-' of hRS were injected i.v. which led to srnaller. though

statistically significant (Pc0.05) increases in both CVBF and DABF. 80th CVBF and

DABF were increased significantly within 3 minutes after the hRS was injected. Peak

flow rates in the CV and DA occurred 6 and 7 minutes after hRS injection and were 4.0

20.29 and 8.1t.49 ml-rnin-'.kg bw-' respectively (Figure 6). The f o m of the flow

responses were similar to those observed following injection of ANG I and ANG II. The

i.v. injection of 1 mg. kg bw" of Pepstatin A completely blocked the increased CVBF

and DABF that had previously followed the injection of hRS (Figure 6). Flow rates

remained at the preinjection level.

Figure 4. Effect of the marnrnalian renin antagonist pepstatin A on the temporal

changes in CVBF ( O ) and DABF ( ) in freshwater North American eels, A.

rostrata. after 2 i.v. injections of 50 ng. kg bw" of [~sn' . val5. G I ~ - A N G I given

before ( time = 55 ) and after ( time = 110 ) the i.v. injection of 1 mg. kg bw" of

antagonist ( time = 100 ). CVBF: ( A to v ) P < 0.05 compared with the

preinjection values ( time = 1 to 50 ). DABF: ( A to ) P < 0.05 compared with

the preinjection values ( time = 1 to 50 ); ANOVA, Duncan's Multiple Range Test.

Values are mean + SEM. n = 5.

Figure 5. Effect of the mammalian renin antagonist pepstatin A on the temporal

changes in CVBF ( O ) and DABF ( - ) in freshwater North American eels, A.

rostrata. after 2 i.v. injections of 50 ng. kg bw-' of [ ~ s n ' . valS]-ANG II given before

( time = 55 ) and after ( time = 110 ) the i-v. injection of 1 mg. kg bw" of

antagonist ( time = 100 ). CVBF: ( A to V ) P c 0.05 compared with the

preiojection values ( time = 1 to 50 ). DABF: ( A to ) P < 0.05 compared with

the preinjection values ( time = 1 to 50 ); ANOVA. Duncan's Multiple Range Test.



changes in CVBF ( O ) and DABF ( = ) in freshwater North American eels, A.

rostrata. after 2 i.v. injections of 150 ng. kg bw-' of hRS given before ( time = 55 )

and after ( time = 1 10 ) the i-v. injection of 1 mg. kg bw-' of antagonist ( time =

100 ). CVBF: ( A to V ) P c 0.05 compared with the preinjection values ( time =

1 to 50 ). DABF: ( A to ) P ç 0.05 compared with the preinjection values (

time = 1 to 50 ); ANOVA, Duncan's Multiple Range Test. Values are mean +

SEM. n = 5.


changes in CVBF ( O ) and DABF ( ) in freshwater North Arnencan eels, A.

rostrata. after 2 i.v. injections of 2.5 mg. kg bw*' of CS-EXT given before ( time =

55 ) and after ( time = 1 10 ) the i.v. injection of 1 mg. kg bw" of antagonist ( time

= 100 ). CVBF: ( A to v ) P c 0.05 compared with the preinjection values ( time

= 1 to 50 ). DABF: ( A to ) P < 0.05 cornpared with the preinjection values (

time = 1 to 50 ); ANOVA. Duncan's Multiple Range Test. Values are mean +

SEM; n = 5.

Figure 7 shows that an extract of 2.5 mg of CS injected i.v. was followed by rapid

and significant increases in both CVBF and DABF. Peak flow rates were 9.5 t 0.24 and

11 -5 20.42 rnl.min?kg bw-' respectively within 10 and 7 minutes after the injection of

CS-EXT. These significant increases in blood flow rates were completed blocked by an

i.v. injection of 1 mg.kg bw" of Pepstatin A. CVBF and DABF remained at preinjection

rates. That implied that the renin-inhibitor had blocked the activity of renin or a renin-like

material contained within the CS,

(D) Captopril and the blood flow responses to ANG I, ANG II, hRS or CS-EXT

Figure 8 illustrates a 266 % increase in CVBF resulting in a peak rate of 10.3 î 0.22

ml.min-'.kg bw"; (Pc0.05) and a concomitant 84 % increase in DABF with a peak rate of

12.9 I 0.39 ml-min ".kg bw"; (P ~0.05) which occurred within approximately 9 minutes

after an i.v. injection of 50 ng-kg bw" of ANG 1. An i.v. injection of 1 rng.kg bw-' of

Captopril blocked completely the blood flow responses to a second i.v. injection of 50

ng.kg bw-' of ANG I (Figure 8). The expetiment was repeated using ANG II instead of

ANG 1. Figure 9 shows that. after the first i.v. injection of 50 ng of ANG II, the CVBF and

DABF rose rapidly and peaked at 10.6 20.16 (278% increase) and 12.3 1-49 ml-min-'.kg

bw" (75% increase). respectively within 8 and 10 minutes, then subsided. The i.v.

injection of 1 mg-kg bw-' of Captopril did not block the flow responses to a second i-v.

injection of 50 ng of ANG II because the angiotensin-converting enzyme (ACE) was

bypassed (Figure 9). The onset of peak flow rates occurred with 7 (CVBF) and 10

(DABF) minutes after the injection of peptide: the peak ffows were 10.5 f 0.19 (275%

increase) and 12.1 2 0.44 ml.rnin-'.kg bw" (73% increase) for CVBF and DABF (Figure

9 ).

Figure 8. Effect of the mammalian AC€ antagonist captopril on the temporal

changes in CVBF ( 0 ) and DABF ( ) in freshwater North American eels, A.

rostrata. after 2 i.v. injections of 50 ng. kg bw" of [ ~ s n ' , val5, GIYI]-ANG I given

before ( time = 55 ) and after ( time = 110 ) the i.v. injection of 1 mg. kg bw" of

antagonist ( time = 100 ). CVBF: ( A to V ) P < 0.05 compared with the

preinjection values ( time = 1 to 50 ). DABF: ( A to ) P c 0.05 cornpared wlh

the preinjection values ( time = 1 to 50 ); ANOVA, Duncan's Multiple Range Test.


Blood flow rate ( ml. min-'. kg bw-' ) 4

CU O 0, O ru 4

Figure 9. Effect of the mammalian AC€ antagonist captopril on the temporal

changes in CVBF ( 0 ) and DABF ( ) in freshwater North American eels. A.

rostrata. after 2 i.v. injections of 50 ng. kg bw-' of [ ~ s n ' , V~I~]-ANG II given

before ( time = 55 ) and after ( time = 110 ) the i.v. injection of 1 mg. kg bw*' of

antagonist ( time = 100 ). CVBF: ( A to V ) P < 0.05 cornpared with the

preinjection values ( time = 1 to 50 ). DABF: ( A to ) P c 0.05 compared with


Values are mean + SEM, n = 5.

0.5 ml saline

I ANG II

(50 ngkg") I A

ANG II (50 ng. kg")

Time (min)

Figure 10. Effect of the marnrnalian AC€ antagonist captopril on the temporal

changes in CVBF ( O ) and DABF ( = ) in freshwater North American eels. A.

rostrata. after 2 i.v. injections of 150 ng. kg bw-' of hRS given before ( tirne = 55

) and after ( tirne = 110 ) the i.v. injection of 1 mg. kg bw-' of antagonist ( time =

100 ). CVBF: ( A to V ) P c 0.05 compared with the preinjection values ( time =

1 to 50 ). DABF: ( A to ) P < 0.05 compared with the preinjection values (

time = 1 to 50 ); ANOVA. Duncan's Multiple Range Test. Values are mean +

SEM. n = 5.

Figure 11. Effect of the mammalian angiotensintonverting enzyme (ACE)

antagonist captopnl on the temporal changes in CVBF ( 0 ) and DABF ( ) in

freshwater North American eels, A. rostrata. after 2 i.v. injections of 2.5 mg. kg

bw" of CS-EXT given before ( tirne = 55 ) and after ( time = 110 ) the i.v. injection

of 1 mg. kg bw-' of antagonist ( time = 100 ). CVBF: ( A to v ) P < 0.05

compared with the preinjection values ( time = 1 to 50 ). DABF: ( A to ) P c

0.05 compared with the preinjection values ( time = 1 to 50 ); ANOVA. Duncan's

Multiple Range Test. Values are mean + SEM. n = 5.

0.5 ml saline

CS-EXT Captopril CS-EXT (1 rng.kgm') (2.5 mg.kg")

Time (min)

Figure 10 shows that there was a modest but significant increase (P<O.OS)in both CVBF

(47%) and DABF (20%) following an i.v. injection of 150 ng.kg bw-' of hRS (human

renin substrate), as observed in the Pepstatin A experiment (see Figure 6). BFR in the

CV peaked at 4.1 ~0 .31 rnl.min".kg bw-' and in the DA at 8.4 20.46 ml-min-'.kg bw" 8

-and 6 min after the injection of hRS. However if the eel was given an i.v. injection of 1

mg-kg bw" of Captopfil before the second dose of hRS, the flow responses were

abolished (Figure 10). In the subsequent . but related experiment, an eet was given an

i.v. injection of an extract of 2.5 mg of fresh CS (Figure 11). Peak flow rates in the CV

and DA were 9.2 t 0.26 and 11.6 I 0.44 ml.min-'.kg bw-' amounting to increases of

230% and 66% respectively. These peaks occurred about 9 min after injection of the

CS-EXT (Figure 11) whereas CVBF and DABF remained above baseline rates for

about 32 and 24 min. An i.v. injection of 1 mg-kg bw" of Captopnl was followed by a

second injection of an extract of 2.5 mg of fresh CS extract, but the flow response was

completely abolished by the drug (Figure 1 1 ).

(E) [Sar', IIe8+ANG II (Sarile) and the blood t7ow responses to ANG II or CS-EX7

Figure 12 shows that both CVBF and DABF increased significantly after an i-v. injection

of 50 ng.kg bw" of ANG II reaching peak flows of 10.3t0.21 and 11 -8 *.49 ml.min-l .kg

bw" at 7 and 10 minutes after injection of the peptide. CVBF and DABF remained

elevated for approximately 33 and 23 minutes respectively, before retuming to pre-

injection baseline rates (Figure 12). Eels were then injected i.v. with 50 pg-kg bw-' of

Sarile and. 10 minutes later. by a further 50 ng.kg bw" of ANG II. There was no

Figure 12. Effect of the mamrnalian ANG II receptor antagonist Sarile on the

temporal changes in CVBF ( 0) and DABF ( = ) in freshwater North American

eels, A. rostrafa. afier 2 i.v. injections of 50 ng. kg bw" of [ ~ s n ' , V~I~]-ANG II

given before ( time = 55 ) and after ( time = 110 ) the i.v. injection of 50 ug. kg

bw-' of antagonist ( time = 100 ). CVBF: ( A to V ) P c 0.05 compared with the




0.5 ml saline

I

ANG I I Sarile ANG Il

(50 ng.kgm') (50 ug.kgS')

Time (min)

Figure 13. Effect of the mammalian ANG II receptor antagonist [ ~ a r ' , IJ~*]-ANG II

(Sarile) on the temporal changes in CVBF ( O ) and DABF ( = ) in freshwater

North American eels. A. rostrata. after 2 i.v. injections of 2.5 mg. kg bw-' of CS-

EXT given before ( time = 55 ) and after ( time = I l O ) the i.v. injection of 50 ug.

Kg bw" of antagonist ( time = 100 ). CVBF: ( A to V ) P c 0.05 compared with

the preinjection values ( time = 1 to 50 ). DABF: ( A to ) P < 0.05 compared

with the preinjection values ( time = 1 to 50 ); ANOVA. Duncan's Multiple Range

Test. Vatues are mean + SEM. n = 5.

0.5 ml saline

I CS-EXT

(2.5 mg.kgS')

I

Time (min)

subsequent increase in either CVBF or DABF which showed that Sarile had blocked

completely the fiow responses to ANG II (Figure 12).

Figure 13 illustrates an experiment in which freshwater eels are first injected i.v.

with an extract of 2.5 mg of fresh CS. After about 10 min. CVBF had increased to peak

rates of 9.5i0.26 (241% increase) and DABF to 11.6 1.47 ml.min?kg bw-' (66%

increase). CVBF and DABF remained elevated for about 33 min and 29 min before

subsiding to the pre-injection rates (Figure 13). There followed an i.v. injection of 50

pg.kg bw" of Sarile which blocked completely the subsequent flow response to a

second injection of extract of 2.5 mg of fresh CS (Figure 13).

(9 Losartan and the blood flow responses to A NG II or CS-EXT

An injection of 50 ng.kg bw" of ANG II was again followed by increased flows in the CV

and DA. The peak blood flow rate in the CV was 10.1 t 0.08 ml-min-'.kg bw" and in the

DA 12.4 2 0.49 ml-min-'.kg bw" amounting to increases of 263% and 77% respectively

compared with pre-injection flows (Figure 14). CVBF remained above baseline for 28

min; DABF for 23 min. The subsequent injection of the mammalian AT, blocker losartan

(5 mg-kg bw-' i-v.) only partially inhibited the flow responses to a second injection of 50

ng.kg bw" of ANG II. The peak CVBF was reduced to 5.9 2 0.29 ml-min-'.kg bw-' (1 12%

increase above baseline) and the D A M to 9.0 t 0.18 ml.min".kg bw-' (29% above

baseline). Here the peak response in the CV took 8 min to develop; in the DA , 10 min

to develop. There was a difference in the duration of the increased flow rates. CVBF

and DABF remained elevated for 27 min and 16 min respectively before returning to

Figure 14. Effect of the mammalian ATi receptor antagonist losartan on the

temporal changes in CVBF ( O ) and DABF ( I ) in freshwater North American

eels, A. rostrata. after 2 i.v. injections of 50 ng. kg bw" of ( ~ s n ' . valS]-ANG II

given before ( time = 55 ) and after ( time = I l 0 ) the i.v. injection of 5 mg. kg bw-

1 of antagonist ( time = 100 ). CVBF: ( A to V ) P c 0.05 compared with the




ANG II Losartan (50 ng.kg") 0.5 ml

saline ANG II

Time (min)

Figure 15. Effect of the mammalhn AT* receptor antagonist losartan on the

temporal changes in CVBF ( 0 ) and DABF ( ) in freshwater North Amencan

eels, A. rostrata. after 2 i-v. injections of 2.5 mg. kg bw" of CS-EXT given before

( time = 55 ) and after ( tirne = 110 ) the i.v. injection of 5 mg. kg bw-' of





baseline pre-injection rates. These experirnents showed that losartan was not a

completely effective AT, inhibitor in freshwater eels (Figure 14).

Figure 15 shows that the injection of an extract of 2.5 mg of CS i-v. was followed

by a 244% increase in CVBF (9.6 * 0.26 ml-min-".kg bw-') and a 67% increase in DABF

(1 1 -7 t 0.47 ml-min-'.kg bw-'). CVBF and DABF rernained elevated for 38 min and 24

min respectively before falling to the pre-injection flow rates. An injection of Losartan (5

mg.kg bw-' i.v. ) reduced significantly the response to the second injection of extact of

2.5 mg of CS. CVBF increased to only 5.5 2 0.09 ml-min-'.kg bw" (97 % above baseline)

and DABF increased to only 9.0 + 0.25 ml-min-'.kg bw-' (29% above baseline). This

amounts to a 43% reduction in the flow response in the CV and a 23% reduction in flow

response in the DA (Figure 15).

(G) PD 72331 9 and the blood fiow responses to ANG II or CS-EXT

Figure 16 illustrates the expected increases in CVBF and DABF following the injection

of 50 ng.kg bd i.v. of ANG II. CVBF increased by 267% to 10.3 20.21 ml.min".kg bw-'

and DABF by 74% to 12.2 20.37 ml.min".kg bw-' . BFR remained elevated in the CV for

33 min and in the DA for 23 min before retuming to the pre-injection rates. Treatment

with the mammalian AT, blocker PD 123319 (5 mg.kg bw-' i.v.) reduced the duration

and intensity of the BF responses to a second i.v. injection of 50 ng-kg bw-' of ANG II.

CVBF increased by only 7.2 I 0.35 ml.min".kg bw-1 (159%) and DABF by only 10.3 2

0.28 ml.min".kg bw*' (47%). The flows remained elevated for 33 min and 13 min

respectively (Figure 16).

Figure 16. Effect of the rnamrnalian AT2 receptor antagonist PD123319 on the

temporal changes in CVBF ( O ) and DABF ( ) in freshwater North Amefican

eels. A. rostrata, after 2 i.v. injections of 50 ng. kg bw-' of [ ~ s n ' . V ~ I ~ - A N G II

given before ( time = 55 ) and after ( time = I l 0 ) the i.v. injection of 5 mg.kg bw-'



the preinjection values ( time = 1 to 50 ); ANOVA. Duncan's Muitiple Range Test.

VaIues are rnean + SEM. n = 5.

S A \

Figure 17. Effect of the mammalian AT2 receptor antagonist PD123319 on the

temporal changes in CVBF ( O ) and DABF ( ) in freshwater North American

eels. A. rostrata, after 2 i.v. injections of 2.5 mg. kg bw-' of CS-EXT given before

( time = 55 ) and after ( time = 110 ) the i.v. injection of 5 mg. kg bw-' of

antagonist ( tirne = 100 ). CVBF: ( A to V ) P < 0.05 compared with the




The i.v. injection of an extract of 2.5 mg of CS was followed by significant and

prolonged increases in CVBF and DABF. At its peak. CVBF had increased by 250% to

9.8 t 0.24 ml.min-'.kg bw-' and the DABF had increased by 66% to 1 1 -6 20.43 ml.minm

'.kg bw-'. The BFR in the CV remained elevated for 32 min whereas the BFR in the DA

remained elevated for 24 min before returning to the pre-injection level (Figure 17).

An i-v. injection of 5 mg. kg bw" of the marnrnalian AT, blocker PD 123319

reduced significantly the responses to a second i-v. injection of an extract of 2.5 mg of

fresh CS tissue. CVBF reached a peak rate of 7.1 10.25 rnl.minA.kg bw" (154%

increase) and DABF a peak rate of 9.7 t 0.24 ml.min".kg bw-' (an increase of 39%).

Flow rates in the CV remained elevated for 22 and 16 minutes respectively before

returning to the pre-injection rates (Figure 17). Overall. PD 123319 only partially

blocked the flow responses to components of the RAS.

The effects of ANG I and ANG II on blood fiow

The RAS has been thoroughly examined in over 100 different species of teleost fishes

(Nishimura 1985, Sokabe 8 Ogawa 1974). ANG 1. a decapeptide, has been isolated

and sequenced following extraction from the plasma of chum salrnon, 0. kefa

(Takemoto et al. 1983). goosefish, L. lifulon (Hayashi et al. 1978). Japanese eel, A.

japonica (Hasegawa et al. 1983) and North American eel. A. rostrata (Khosla et al.

1985). ANG I is a biosynthetic intermediate of the RAS and has no known physiological

function (Hirano & Hasegawa. 1984). Angbtensin conveRing enzyme (ACE) which. in

fishes is located primarily in the gills and kidney (Nishimura 1985). rapidly converts

ANG I to ANG II by cleaving a dipeptide from the C-terminal end of the molecule. Thus

formed, ANG II has powerful direct and indirect effects on cardiovascular function in

teleosts as well as higher vertebrates (Olson et al. 1989, Polanc et al. 1990).

ANG II is the principal effector peptide of the RAS in teleost fishes, wherein it exerts

a powerful vasopressor effect (Nishimura 8 Sawyer 1976. Churchill et al. 1979. Zuker 8

Nishimura 1981, Carroll & Opdyke 1982, Perrott & Balment 1990). ANG Il is also known

to increase cardiac output through its chronotropic and inotropic effects which lead to

an increase in arterial blood flow and peripheral vasoconstriction in freshwater North

American eels (Oudit 8 Butler 1995). ANG II also stimulates thirst (Carrick & Balment

1983. Balment & Camck 1985) during adaptation of euryhaline fishes to seawater. ANG

II is converted to ANG Ili by the cleavage of the N-terminal amino acid asparagine (Asn)

and its conversion to a heptapeptide. The conversion is achieved by the action of

peptidases known to be abundant in the kidneys and gills (Olson 1992).

In the present study, approximate physiological doses of the peptides. ranging

from 5 to 50 ng. kg bw" were used. Due to the rapid. peripheral conversion of ANG I to

ANG II (Nishimura 1985). the effects of these peptides on flow were nearly identical.

For instance. a 5 ng. kg bw-l dose of either [Asnl, Val5, GIfl-ANG I or [Asn', Va15]ANG

Il caused a significant increase in venous blood flow, The increase in the rate of arterial

blood flow became significant at the 10 ng. kg bw-' dose (Figure 2). The responses

were higher at the 20 ng. kg bw-' dose and the highest at the 50 ng. kg bw-' dose.

Because the range of doses did not exceed 50 ng. kg bw". a tme curvilinear dose-

response relationship could not be established. However. I expenments show a strong

positive correlation between the dose of peptide delivered and the resultant blood flow

response. These results are consistent with eariier studies that showed that i.v. injection

of [Asn'. Val5. GlyS]-ANG I or [ A d . V~I~]-ANG II were followed by significant pressor

responses in North American eels (Nishimura 8 Sawyer 1976. Nishimura et al. 1978).

Oudit and Butler (1995) were the first investigators to demonstrate that the increase in

arterial blood flow was the main contributor to the ANG il mediated pressor response in

freshwater North American eels. They found that [Asnl, Val5. GIfl-ANG I or [Asn', val5]-

ANG II has stimulatory effects on heart rate and stroke volume which increases artenal

blood flow (cardiac output), and leads subsequently to higher arterial blood pressure-

Although blood pressure was not measured in our experiments. the increase in arterial

blood flow. following i.v. injection of [Asnl. Vals. Glyq-ANG I or [Asnl. Val5]-ANG II may

have also caused an increase in arterial blood pressure.

Figure 1 shows the dorsal aortic and caudal venous calibration curves for three

freshwater eels. All of the regression lines have nearly identical slopes showing the

highly significant positive Iinear correlation between the blood flow rate and

corresponding Doppler shifts. Dorsal aortic and caudal venous blood flows were

-determined by interpolation from these curves. Velocity profiles across the dorsal aorta

and caudal veins (2.8 mm probe) were determined by making small serial increments in

the range output which allowed us to determine the optimal distance at which blood

velocity could be measured. As in earlier experiments (Butler & Oudit 1995, Oudit 8

Butler 1995), both the dorsal aortic and caudal venous flow profiles were parabolic,

indicating that flow was laminar in both vessels. During the experiments, the range was

adjusted so that the peak mean blood flow was always measured. Similar velocity

profiles have been observed in the pulmonary arteries of green turtles, Chelonia mydas

(West 1989) and in the ventral and dorsal aortae of freshwater eels (Butler & Oudit

1995). In summary 1 was fully confident that Doppler flow probe measurements in my

experiments gave valid estimates of blood flow velocities.

The Relationship between cardiac output, dorsal aortic blood pressure and dorsal

aortic and caudal venous blood flows

Butler and Oudit (1995) have studied the relationship between cardiac output (CO),

dorsal aortic blood flow (DABF) and dorsal aortic blood pressure (P,,) in intact

freshwater eels before and after removal of the corpuscles of Stannius (CS). which they

presumed to contain renin or a renin-like substance. Three weeks after removal of the

CS. there followed a 100% increase in estimated branchial shunting wherein the blood

bypassed the gills and flowed directly to the dorsal aorta, and also a 25% decrease in

cardiac output. DABF decreased by about 45% one week after CSX and remained

there for the duration of the experÏment. PD, fell by 15% within four days after the

glands were removed and gradually climbed back into the normal range by the 12<" day.

These findings implied that the CS release a renin or a renin-like substance which led to

further experïments by Oudit and Butler (1995). Their hypothesis was that the end-

product of the corpuscular RAS (CRAS) is ANG II and that this peptide regulates

cardiovascular function in freshwater eels.

CO, DABF and PD, were rneasured simultaneously in conscious freshwater eels

following i-v. injections of a series of graded doses of 25. 50. 100 and 150 ng.kg bw" of

[ A d , Val '1 Angiotensin II (ANG II) (Figure 18). CO increased gradually at all doses but

the increase was disproportionately large at the lower doses (Figure 18A). For example.

25 ng.kg bw-' increased the CO by 4 ml.min-'.kg bw-' whereas a six-fold increase in

dose (1 50 ng-kg bw-') increased the CO by only 8.2 ml.min-'.kg bw? Muscarinic

receptor blockade with atropine potentiated these responses to graded doses of ANG II

implying that cholinergie innervation of the eel heart initially downregulates CO via a

baroreceptor reflex. ANG II also produced a dose-related increase in PD, (Oudit 8 Butler

1995) which has been observed in other studies on freshwater eels (Nishimura et al.

1978, Hirano & Hasegawa 1984). At a dose of 100 ng-kg bw" of ANG II. CO increased

slightly within the first min but leveled off for a further two min suggesting a reflex

inhibition of cardiac frequency. However, there was

Figure 18. Temporal changes in CO (A) [control-( n = 8: a ) and atropine treated

( n = 6; O ) 1. mean PD* (6; n = 6). and R,, (C: n = 6) in freshwater North

Amencan eels Anguilla rostrata. after intravenous injection of 100 ngkg of [~sn ' .

val5]-ANG II. Dorsal aortic blood flow followed the same trend as the changes in

CO. A P < 0.01 compared with the preinjection value (time = O); 6 P < 0.05

compared with atropine treated. Values are mean 2 SE. (Adapted wlh

permission from Oudit and Butler, 1995)

a rebound. as CO climbed rapidly reaching a peak within 9 min and remaining elevated

for 15 min when the experiment ended (Figure 18B). In contrast the P, increased

rapidly from the outset. peaked at 4 min and drifted downward slowly. By 15 min it had

fallen to about 50% of the peak PD,. Peak systemic vascular resistance coincided with

the peak PD; and fell rapidly thereafter so that it was only slightly above baseline for the

last half of the 15 min observation period (Figure 18C). In summary. Oudit and Butler

(1995) have shown that the increased CO due to increased stroke volume and cardiac

frequency are the driving force for the increased P, which follow elevated circulating

levels of [ A d , V a 1 5 ] - ~ ~ G II. Nevertheless. these conclusions have Oeen drawn without

measurements of dorsal aortic blood flows or retuming blood flows through the large

caudal vein. If caudal venous flow increased substantially. then according to the Frank

Starling Law. the increased filling pressure of the eel atrium (end diastolic volume)

would lead to yet a further increase in cardiac output, superimposed on the direct action

of ANG II on heart rate and stroke volume. Thus I have expanded on the work of Butler

and Oudit (1 995) and Oudit and Butler (1995) by measuring the dorsal aortic blood flow

and caudal venous blood flow in freshwater eels following the i.v. injection of a series of

doses 5. 10. 20 and 50 ng-kg bw" of [Asn'. Val5, Glfl-ANG 1. [Asn'. Vaq-ANG II and

[Val4]-ANG III (Figure 2). The amount of hormone, as low as 5 ng. kg bw" of [Asn'.

V~ I~J -ANG II. required to elicit the observed cardiovascular responses. are some of the

Iowest reported to date. Therefore, the measurement of blood flow in response to

peptides. may be useful as a sensitive bioassay for testing elements of the RAS such

as ANG 1. II and III or other vasoactive peptides such as 8-arginine vasotocin.

The effects of ANG 111 on blood flow

Figure 2 shows that DABF increased significantly following injections of 10. 20 and 50

ng of [Asn'. vals. Glfl-ANG I and that al1 four doses increased CVBF. This showed that

peripheral conversion of [Asnl. Val5. GlflANG I to [Asnl. VaITANG II was rapid and

that each dose of this biologically inactive intermediate was as effective as an equal

dose of exogenous ANG II (figure 2). Again. with [Asn'. Van-ANG II a 5 ng-kg bw-'

dose did not significantly increase DABF. These flow responses to doses of [ A d .

val5]-ANG II as low as 5 ng-kg bw-' are well within the physiological range and are the

lowest yet reported to give a cardiovascular response to ANG I or Il, in any species of

fish. In rnammals. ANG III possess only 40% of the pressor activity of ANG II (Dzau et

al. 1988. Hall et al. 1992). Initially, it had been reported that [Asn4]-ANG III has virtually

no pressor activity in Japanese eels. Anguilla japonica (Hirano 8 Hasegawa 1984).

However. Butler and Oudit (1995) have recently shown that 25 ng-kg bw-' of [Asnq-

ANG III increased CO by 23% by way of increasing stroke volume but not heart rate.

Even though 150 ng-kg bw-' increased CO by 47%. there was still no measurable effect

on heart rate. This lack of change in heart rate represented a clear difference in the

cardiac responses to ANG II and ANG III. Peak PD, increased by 25% in response to 25

ng and by 52% in response to 150 ng-kg bw" of ANG III which may have been due to a

combination of an increased CO and peripheral vasoconstriction. The above

observations (Butler & Oudit 1995) were supported by the present study. For example.

Figure 2 shows that even though a 20 ng. kg bw-' i.v. injection of ANG III increased

significantly CVBF (30%) it had no measurable effect on DABF. At the higher dose of

50 ng-kg bw-' of val4]-ANG III . CVBF increased by 52% and DABF by 24%. Therefore

DABF was less affected. My observations may show that pal4]- ANG III diffen from

[Asn'. val5. Glfl- ANG I and [Asnl. Val5]-ANG II insofar as the hormone increases CO

slightly through its direct effect on stroke volume but not on cardiac frequency. The

increase in PD, is achieved by peripheral vasoconstriction.

The effect of [Asnl. Val5. Glfl-ANG 1. [Asn'. ValSI-ANG II and [ Va14]-ANG Ill on

blood fiow were tested in North American eels. We have shown that i-v. injections of

ANG 1, ANG II or ANG Ill. cause dose-dependent increases in artenal (dorsal aorta) and

venous (caudal vein) blood flows. These results augment the findings of earlier studies

of the effect of these peptides on the arterial blood pressure and blood flow in some

fishes.

Evidence for a renin or renin-like enzyme in the CS: flow responses to CS-EXT

and hRS

Now that the experirnental set-up has been shown to be sensitive to angiotensins and

that CVBF and DABF responses to as little as 5-10 ng-kg bw-' of [Asnl, v~IT-ANG II.

one rnay proceed with the next step. That is to show that the CS contain renin or a

renin-like substance. Figure 3 illustrates three experiments that are related to this

hypothesis. In order to exclude the possibility that the posterior kidney contains

substantial or measurable arnounts of renin, we tested the flow responses in the DA

and CV following the i-v. injection of 5.0 mg of a saline extract of the posterior or

functional kidney. Since this region contains the majority of the filtering glomenili, and

therefore the bulk of the juxtaglomerular cells containing renin (Capreol and Sutherland

1968) one might expect a pressor or flow response from the extract- However, there

was no measurable response to this extract but there was hormonal activity in the CS

extracts.

This is the f int study to demonstrate that approxirnate physiological doses of an

extract containing 0.50, 1.25 or 2.50 mg. Kg bw-' of fresh CS can cause immediate and

sustained, dose-dependent increases in artenal and venous blood flows. Only the

lowest dose (0.50 mg. kg bw-'), equivalent to one-tenth of a corpuscle per kg bw. failed

to elicit a significant flow response. whereas a 1.25 or 2.50 mg. kg bw' dose of CS

increased arterial and venous blood fiows well beyond baseline levels (Figure 3). The

results of my study strongly support earlier findings on the pressor effects of the CS.

Extracts from the CS of four different species of teleosts (A. anguilla, C. carpio.

C. auratus. L. litulon) were al1 found to have significant pressor effects in anaesthetized

rats (Chester-Jones et al. 1966. Sokabe et al. 1970). Surgical removal of the CS

(stanniectomy) in European eels, A. anguilla and North American eels, A. rostrata is

followed by a rapid decline in arterial blood pressure (Chester-Jones et al. 1966, Bailey

& Fenwick 1975). In a recent study. as much as a 15% decline in dorsal aortic blood

pressure and a 45% decline in dorsal aortic blood fiow were reported in the North

American eel following stanniectomy (Butler et al. 1995). In stanniectomized eels, the

blood pressure depression was corrected by intravenous injections of extracts of eel CS

(Chester-Jones et a/. 1966). However, there are no published reports on the effect of

CS extracts on dorsal aortic or caudal venous blood flow rates. Rernarkably. the

duration and the intensity of the response to just 2.50 mg. kg bw" of CS is equivalent to

a 50 ng. kg bw-' injection of [Asn'. Val5, Glfl-ANG I or [Asnl, Va15]ANG II. Our

experiments cleariy show that the CS contain a vasoactive principle that is a potent

stimulator of arterial and venous bfood flow, in the intact North American eel.

A saline extract of 0.5 mg .kg bw-' of CS-EXT failed to change DABF or CVBF.

but an i-v. injection of 1.25 mg of CS-EXT was followed by a 127% increase in CVBF

and a 45% increase in DABF. 60th of these increases were statistically significant

(Pc0.05) compared with saline-injected control values (Figure 3). Even greater

responses were observed following the injection of an extract from 2.5 mg CS. kg bwl.

CVBF increased by 237% and DABF by 64%. both fiows being significantly greater

than both the saline injected controls and in the 1.25 mg group. This observation.

added to the discovery by Chester Jones et al. (1966) that extracts of freshwater

European eel. A. anguilla. CS contained a renin-like pressor substance. strengthens rny

hypothesis that the CS contain significant renin-like activity in teleost fishes. Finally, l

injected three doses (50. 100 and 150 ng.kg bw-' i.v.) of human renin substrate (hRS) to

show if endogenous eel renin from either the posterior kidney JG cells andfor the CS

cleave the decapeptide ANG I from this substrate. It would be rapidly converted to ANG

II by endogenous ACE and one rnight expect to observe changes in CVBF and DABF

similar to those shown in Figure 2 in response to injected [Asn'. Val5. Glfl-ANG I and

[Asn'. Val5]-ANG II. Figure 3 shows that at the intemediate dose there was a

statistically significant (P<0.05) 38% increase in CVBF but no change in DABF. At the

highest dose of 150 mg-kg bw-l i-v.. both the CVBF (16%) and DABF (42%) increased

significantly. Apparently hRS was an adequate but not quantitatively strong substrate

for endogenous eel renin (Figure 3).

The response to human renin substrate [Asp', Iles. Hisq (hRS) has not been

previously tested in freshwater North American eels. It had been reported that kidney

extract from the eels can fom angiotensin from synthetic hRS as well as fowl renin-

substrate (Nishimura 1980). The results of our study suggest that in the eel, the

conversion of hRS to angiotensins can also occur in vivo. It should be considered that

in mammals, the reaction of renin on renin-substrate shows strong species specificity

(Schaffenburg et al. 1960, Grill et al. 1972). In our case. the reaction between eel renin

and heterologous substrate may not have proceeded with great efficiency, resulting in

the inadequate production of angiotensins and hence the observed weak flow

responses.

Inhibition of putative renin in eel CS-EXT and of hRS by Pepstatin A

Flow responses to extracts of CS were inhibited by prior i.v. injections of 1 mg. kg bw-'

of pepstatin A. This implied that the CS contained 'renin" (Figure 7). Pepstatin A is a

bacterially derived pentapetide (Umezawa et al. 1970) which is a highly effective,

cornpetitive inhibitor of acid proteases such as human pepsin, gastricin, cathespin D

(Marciniszyn et al. 1976) and human renin (Gross et al. 1977). It has been suggested

that Pepstatin A is unsatisfactory for use in studies of the RAS because it lacks

specificity for renin (Gauten et al. 1984. Haber 1985). However. of al1 the acid

proteases. renin is the only enzyme that can fomi ANG I at a significant rate. Pepstatin

A decreases renin activity ( P M ) by at least 80% (Hidaka et al. 1985). The intravenous

injection or constant infusion of Pepstatin A reduced blood pressure and blocked the

pressor effects of exogenous renin in rats (Evin et al. 1978. Miyazaki et al. 1979,

Antonaccio 1982).

This is the first expenmental test of the effects of pepstatin A in any species of

fish. The effects of pepstatin A in freshwater eels were found to be analogous to its

effects in mammals (Figure 6). revealing the highly consewed nature of the RAS. We

showed that pepstatin A can inhibit renin-activity in vivo since the flow response to hRS.

the renin substrate. was also abolished (Figure 6). Furthemore. the inhibitory effect of

pepstatin A was shown to be renin-specific. since the flow response to i.v. injections of

[Asn'. Val5, GIfl-ANG I (Figure 4) were not prevented by prior administration of

Pepstatin A. This result was expected since the conversion of [Asn'. Vals. Glfl-ANG I

to [Asn'. Val)-ANG II the active peptide. depends only upon AC€ which is not blocked

by Pepstatin A. Finally. Pepstatin A had no measurable effect on CVBF or DABF

responses to ANG II (Figure 5) because no enzymatic conversion of the peptide takes

place in the RAS. ANG II is therefore able to act with full force.

The functional connection between the CS and the RAS has been investigated

previously using a variety of different experimental approaches. In each case. the

investigators concluded that the CS contain a renin-like pressor that is capable of

forming ANG 1 . It is known that extracts made from the CS of freshwater eels contain a

powerful pressor that has many of the physical and functional properties of mammalian

renin (Chester-Jones 1966). Studies show that the CS from the Japanese eel (Ogawa

et al. 1982), the chum salmon (Takernoto et al. 1983) and the goosefish (Hasegawa et

al. 1984) can produce ANG I when it is incubated with homologous plasma. In our

study, we compared the fiow response to CS-EXT in intact anirnals with the same

response under the condition of renin-inhibition, carried out by a 1 mg. kg bw-' injection

of pepstatin A. For instance (Fig. 7). in the unblocked state, blood flows in the dorsal

aorta and the caudal vein becornes significantly elevated for 24 to 33 min following CS-

EXT injection. compared to blood flow during the preceding (55 min) vehicle injected

period (Duncan's Multiple Range Test. p < 0.05). However. in the blocked state, CS-

EXT failed to elicit any significant increase in flow. Therefore. we clearly demonstrated

that renin activity underscores the flow and presumably the pressor response to CS-

EXT.

Depending on the species examined. the total renin activity of teleost CS is

estirnated to be only 0.1 to 0.7 % that of the kidney (Sokabe et al. 1970). However. I

showed that the renin-like activity found in just 1.25 mg. kg bw-' of CS-EXT (equivalent

to approximately one quarter of a CS. kg bw-'), could elicit significant increases in

arterial and venous blood flow (Figure 3). One may consider the fact that the posterior

or functional kidney of eels contains relatively few large nephrons (Audige 1910).

Therefore one may hypothesize that the actual number of putative renin-secreting

granulated cells may be far greater in the two functioning CS, where their nurnber

exceeds 90% of the total cell content. Of particular relevance is the fact that compared

to renal renin. isorenins found in both rat brain and hog spleen. exhibit a 1000-fold

greater sensitivity to the inhibitory action of Pepstatin A (Hackenthal et al. 1978). On the

basis of rny study. the CS of North American eels may also contain isorenins that are

highly sensitive to Pepstatin A. and are capable of forming angiotensins with a higher

than expected efficiency.

Blood flow responses to putative renin in eel CS-EXT and to hRS:

(A) Inhibition of the second-step endogenous conversion of ANG 1 to ANG II with

cap topril

Captopril is a potent inhibitor of angiotensin-converting enzyme (ACE). and blocks the

-enzymatic conversion of ANG I to ANG II (Ondetti et al. 1977). In my study. Captopril

completely blocked the flow responses to i-v. injections of CS-UCT (Figure 11) . hRS

(Figure 10) and ANG 1 (Figure 9) but not to ANG II (Figure 8). as would be predicted-

In ma mmals, Captoprif reduces blood pressure and blocks the pressor response

to i.v. injections of ANG 1. without affecting the response to ANG II (Ferguson et al.

1977. Gavaras et al. 1978. Rotmensch et al. 1988). In the rainbow trout. 0. Mykiss,

Captopril inhibits ACE-activity which is located primarily in the gills- As a result. there

follows a significant reduction in dorsal aortic blood pressure presumably because of

decreased circulating levels of ANG II (Lipske et al. 1987). Captopril and its natural

analogue, teprotide (SQ 20881). block the pressor effects of exogenous goosefish

[Asn'. Val5. Hisq-ANG I or chum salmon [Asn', Val5. TyflANG 1. in unconscious-

vagotomized rats (Nakajima et al. 1971). Similarly. SQ 20881 and presumably Captopril

can inhibit the pressor response to ANG I in North Arnerican eels but not the pressor

response to ANG II (Nishimura et al. 1978). Such studies confirrn not only the presence

of ACE in North American eels. but also the absolute requirement of ACE-activation in

the fl ow and pressor responses to ANG I and its precursor. hRS.

In mammals, ACE catalyzes not only the formation of ANG II, but also the

degradation of bradykinins (Erdos 1977). Bradykinins are powerful vasodepressors that

act by stimulating the production of arachidonic acid metabolites, nitric oxide and

endothelium-derived hyperpotarized factor (Vanhoutte 1989). Therefore, ACE inhibition

should restrict the formation of ANG II. and promote the accumulation of bradykinins.

Studies show that bradykinins may also be present in teleosts (Olson et al. 1997),

although their function is unclear. For instance, kinin-like substances have been

identified in the carp, Cyprinus carpio (lnouye et al. 1961) and the rainbow trout (Dunn

& Perks 1975). The occurrence of kinin-like substances in other teleost species, such -

as the North American eel, cannot be discounted. As stated above, my study shows

that the ACE inhibitor Captopril can abolish the blood flow response to CS-EXT, hRS

and ANG 1 . Under these conditions, when AC€ is inhibited, kinin-like substances may

accumulate in tissues and possibly affect blood flow and pressure. If so, one should

interpret the flow responses with caution and consider that the changes in fiow in

response to Captopril may not be caused only by a reduction in blood levels of ANG II.

In my study, one may Iargely discount any interference from putative kinins because the

extent and duration of the fiow responses to [Asn', Va15]ANG II are similar before

treatment with Pepstatin A (Figure 5). before treatrnent with Captopni (Figure 9) and

after treatment with Captopril (Figure 9).

(B) Effects of the AT, and AT, receptor antagonist Sarile

In this study, i-v. injection of 50 ug.kg bw-' of Sarile. a potent ANG II- receptor

antagonist (Yamamoto et al. 1972). abolished the increased CVBF and DABF which

followed the i.v. injection of 2.5 mg. kg bw-' of CS-EXT (Figure 13). This implies that

renin-like activity is contained within the CS-EXT's. and had ultimately generated

endogenous [Asn', ValS]-ANG II which in turn, increased CVBF and DABF. When i.v.

injection of the same amount of CS-EXT was preceded by mammalian AT, and AT,

receptor blocken. the extract failed to generate the earfier increases in CVBF and

DABF (Figure 13). The next experiment (Figure 12) showed that the amplitude and

duration of the increased CVBF and DABF responses to ANG Il almost matched the

responses to CS-EXT (Figure 13). If an i-v. injection of 50 ug.kg bw-' of Sarile was given

before the second injection of [Asnl. Val)-ANG II. the flow responses were blocked

completely (Figure 12). Sarile was fully effective as an ANG II inhibitor in these

freshwater eels. a finding that is in accordance with earlier observations showing that

Sarile inhibits the pressor effects of ANG II in rats and canines (Johnson et al. 1972.

Turker et al. 1972). Moreover. Sarile can inhibit the other actions of ANG II. including

the stimulation of aldosterone synthesis and the myotrophic effects on vascular smooth

muscle (Johnson et al. 1973, Hall et al. 1974). Through the use of radioimmunoassays.

Sarile has been shown to bind indiscriminately to both AT, and AT, recepton

(Timmermans et al. 1993).

Sarile also blocks the pressor responses to ANG II in birds, reptiles. amphibians

and fishes other than eels. For example. Sarile can block the pressor response to ANG

II in Pekin ducks. Anas platyrhynchos (Butler et al. 1998) and chickens. Gallus

domesticus (Moore et al. 1981. Nakamura et al. 1982). Moreover. a clear inhibitory

effect of Sarile on the pressor response to ANG II has been observed in Western

painted turtles, Pseudemys scripta elegans (Zehr et al. 1981) and in bullfrogs. Rana

catesbeiana (Harper et al. 1985).

In some cases. ANG II receptors which were unaffected by nonpeptide

antagonists such as Losaratan or PD 123319 were fully blocked by peptide antagonists

such as Sarile. For example, ANG II receptors in the adrenal gland of Pekin ducks. A.

platyrhynchos (Gray et al- 1989) and the heart of African clawed toads. Xenopus laevis

(Bergsma et al. 1993) bind Sarile but not other known ANG II antagonists such as

Losaratan and PD 123319. On the other hand, my experirnents have shown that in A.

rostrata, ANG II receptors respond to both Sarile (Figures 12 and 13) and nonpeptide

antagonists such as the AT, receptor blocker Losartan (Figures 14 and 15) and the AT,

blocker PD1 2331 9 (Figures 16 and 17). The latter experiments with nonpeptide ANG II

receptor antagonists reveal further that these receptors may represent two distinct

subtypes, similar to AT, and AT, receptors in mammals.

(C ) Effects of the AT, receptor antagonist Losartan and the AT, receptor

antagonist PD 123319

My experiments are the first to show that i-v. injection of the synthetic nonpeptide ANG

II receptor antagonists. Losartan and PD123319 have inhibitory effects on the CVBF

and DABF responses to CS-EXT (Figure 15 and 17) and ANG II (Figure 14 and 16) in

freshwater eels. Eariier studies on rat and bovine tissues have shown that Losartan and

PD1 2331 9 bind discriminately to 2 subpopulations of ANG II receptors. designated as

AT, (Losartan) and AT, (PD1 23319) receptors (Chang et al. 1990. Bumpus et al. 1991 ).

AT, receptors are found in the heart, vascular tissues, adrenal cortex, renal tubules and

the circurnventricular organs of the brain (Chiu et al. 1989. Herblin et al. 1991 ). As such.

these receptors mediate al1 of the classical responses to ANG II including

vasoconstriction, blood pressure elevation, aldosterone release, renal tubule sodium

reabsorption, thirst. sodium appetite, as well as hyperplesia and hypertrophy of vascular

and cardiac smooth muscle (Timmermans 1993). All of the aforementiohd central and

peripheral actions of ANG II are inhibited by the AT, receptor antagonist Losartan, (see

reviews by Griendling et al. 1994. Messerli et ai. 1996). In studies on rats and dogs, i.v.

injections of Losartan blocked the pressor effects of ANG If, while the pressor response

to other hormones such as norepinephrine or vasopressin were unaffected (Chiu et al.

1990, Wong et al. 1991 ).

AT, receptors on the other hand. are less prevalent and their function is

unknown. In rats. the highest concentrations of AT, receptors occur in the adrenal

medulla. utenis. thalamus and the developing fetus. though their numbers dirninish

dramatically post-partum (Smith et ai. 1992. Viswanathan et al. 1991). Inhibition of AT,

receptors with PD1 2331 9, PD1 231 77 or CGP42112 does not affect the responses to

ANG II, including pressor responses (Wong et al. 1990. Dudley et ai. 1990, Griendling

et al. 1994).

The results of our study show that blood flow responses to ANG II in freshwater

eels. may be mediated concurrently by AT, receptors and AT, receptors. The fint

experiment showed that the increase in CVBF and DABF which followed the i.v.

injection of CS-EXT (Figure 15) were only partially blocked by an i-v. injection of 5

mg-kg bw" of the mammalian AT, antagonist losartan (Figure 15). The reduced flow

response was not due to a failure of the CS-EX1 initiated [Asnl, Val)-ANG II generating

system (Figure 15) because in a second expenment using [Asn'. Val?-ANG II. the

result was much the same. Figure 14 shows that an i.v. injection of 50 ng-kg bw-' of

[Asnl. v~I~]-ANG II gave a typical and clear increase in both CVBF and DABF which

again. as in the CS-EXT experiment was only partially blocked with Losartan.

Since only partial blockade of the flow responses to [Asnl. VaqANG II was

achieved with the marnmalian AT, antagonist Losartan, it was important to repeat the

experiment using the AT, antagonist PD 123319. Figure 17 shows that eel CVBF and

DABF both increased, as before (Figure 15) following an i.v. injection of 2.5 mg of fresh

CS. This increase was diminished partially if the eels were given an i.v. injection of 5

mg-kg bw" of the AT, antagonist PD 123319 before the subsequent injection of CS-

EXT (Figure 17). This result implied that the CS extract generated endogenous ANG II.

In the second experiment (Figure 17). 1 observed that. as with Losartan, PD 123319

only partially blocked the fiow responses to ANG II (Figure 17). 1 concluded that both

AT, and AT, receptors may collaborate to directly andlor indirectly regulate CVBF and

DABF. Although AT, or AT, inhibition alone. failed to abolish the DABF and CVBF

responses to i-v. injections of ANG II or CS-EXT. their combined inhibitory effect on

DABF and CVBF is close to 100%. Therefore, one can argue that AT, and AT,

receptors effectively rnediate al1 of the DABF and CVBF responses to ANG II or CS-

EXT.

My studies augment previous findings on the characterization of ANG II

receptors in teleost fishes. In the rainbow trout. 0. Mykiss, ANG II receptors have been

found in many tissues including the heart, gills, kidney. brain and the adrenocortical

homologue (Cobb et al. 1992). Of particular relevance to my investigation is the

identification of AT,-like receptors in the liver. gill. kidney and intestines of freshwater

European eels, A. anguilla. In addition to the AT,-like receptors, in these eels, there is

evidence for a second receptor subtype in the liver (Manigliante et al. 1994. 1995).

Similarly, AT,-like receptors have been identified in toadfish vascular smooth muscle

(Nishimura & Qin 1994). The existence of multiple ANG II receptor subtypes in the eel

is strongly supported by the results of our study which show that the blood flow

response, and presumably the pressor response to [Asnl. Val7-ANG II (Butler et al.

1995), is rnediated by two subtypes of ANG II recepton. akin to mammalian AT, and an

AT, receptors.

Alternatively. ANG II receptors in North American eels rnay represent a unique

receptor subtype that has characteristics of both AT, and AT, receptors. Experirnental

evidence suggests that ANG II receptor heterogeneity is prevalent among lower

vertebrates. Receptors for ANG II have been well characten'zed in birds and

amphibians. where they mediate rnany of the classical responses to ANG II. including

pressor effects (see review by Nishimura 1980). Avian ANG II recepton may also differ

from mammalian AT, and AT, receptors. since the predicted effects of nonpeptide ANG

II receptor antagonists on the actions of ANG II. are not observed. In Pekin ducks. A.

platyrhynchos, i.v. injections of both losartan and PD123319 failed to block either the

pressor response. or the attenuation of nasal salt gland secretion following i.v. infusion .

of ANG II (Butler et al. 1998). Furthermore. studies on the domestic fowl. G.

domesticus, showed that neither losartan nor PD123319 inhibit the actions of ANG II on

isofated blood vessels and adrenal tissues (Le Noble et al. 1991, Hasegawa et al. 1993.

Gray et al. 1989). In clawed toads. Xenopus laevis ANG II receptors are found in the

heart, kidney, interrenais and ovarian follicles. They have a low affinity for Losartan

(Kloas et al. 1992. Sandberg et al. 1990. Sandberg et al. 1991 ). Thus the presence of

novel ANG II receptors that mediate the flow and pressor effects of ANG II in North

American eels would not be inconsistent with evolutionary trends.

Figure 1 9. Stepwise pharrnacological inhibition of the blood flow responses to peptide

components of the eef RAS, incuding the possible rote of the CS.

DABF (Dorsal Aortic Blood Flow), CO (Cardiac Output)

CVBF (Caudal Venous Blood Flow), VR (Venous Retum)

(1) ANGIOTENSINOGEN (RENIN-SUBSTRATE)

Renin-Activity 1 +.------ Renin inhibition (CS-EXT) ( Pepstatin A)

ACE I + - - O - O - AC€ inhibition (gills) (Cap topril)

AT, antagonism- - - - - + A T2 antagonism (Losartan) (PD12331 9)

*Nai 1 Receptors

lncreased DABF(C0) and CVBF(VR)

.-_--O Receptor antagonisrn (Sarile)

SUMMARY

1) DABF and CVBF increased in freshwater North Amencan eels, A. mstrata, in a

dose-dependent manner following i.v. injection of 5. 10. 20 and 50 ng. kg bw" [Asnl.

Vals. Glfl-ANG I or [ A d . Val5]-ANG II. The effects of [Asn'. Vals. Glfl-ANG I and

[Asnl. va15]4NG II on DABF and CVBF were identical, the minimum effective dose was

5 ng. kg bw-'.

2) Physiological doses of pal?-ANG 111 (5. 10. 20 and 50 ng. kg bw-') increased DABF

and CVBF in a dose-dependent manner. The minimum effective dose of pal4]-ANG III

was 10 ng. kg bw?

3) lntravenous injection of 0.5. 1.25 or 2.5 mg. kg bw-' CS-EXT caused immediate and

sustained dose-dependent increases in DABF and CVBF. Similar increases in DABF

and CVBF was observed following the i.v. injection of 50. 100 or 150 ng. kg bw-' hRS.

The DABF and CVBF responses to the 2.50 mg. kg bw" injection of CS-EXT were

comparable to the responses to 50 ng. kg bw-' of [Asnl. Val5. Glfl-ANG I or [Asnl.

Val5]-ANG II.

4) Renin-like activity mediates the DABF and CVBF responses to hRS and CS-EXT.

The blood flow responses to 150 ng. kg bw-' hRS or 2.5 mg. kg bw-' CS-EXT were

completely blocked by a prior injection of 1 mg. kg bw" of the mammalian renin

inhibitor. Pepstatin A. Pepstatin A did not affect the Row responses to 50 ng. kg bw-' of

[Asn'. Vals. Glfl-ANG I or [Asn'. ValS]ANG II.

5) The DAEF and CVBF responses to [Asnl, Val5. Glfl-ANG 1. hRS or CS-EXT are

ACE-dependent. 1 mg. kg bw-' of the mammalian ACE-inhibitor, Captopfil completely

abolished the flow responses to 50 ng. kg bw" [Asnl. Val5, Glfl-ANG I , 150 ng. kg bw-

' hRS or 2.5 mg. kg bw-' CS-EXT. Captopnl did not affect the flow responses to 50 ng.

kg bw-' of [Asn'. ValS]-ANG II.

6) The marnmalian AT, and AT, receptor antagonist [Sar'. Ile7-ANG II (Sarile) is a

potent in hibitor of the DABF and CVBF responses to [Asn'. Val5]-ANG II or CS-EXT. 50

ug. kg bw-' Sarile completely blocked the flow responses to 50 ng. kg bw-' of [Asnl.

Val5]-ANG II or 2.5 mg. kg bw-' CS-EXT.

7) The CS of freshwater North American eels. A. rostrata, contain a potent renin-like

pressor substance which participates in the synthesis of ANG II. in conjunction with

other endogenous elements of the eel RAS

8) Losartan. the mammalian AT, receptor antagonist or PD1 233 19. the mammalian

AT, receptor antagonist partially blocks the DABF and CVBF responses to 50 ng. kg

bw-' of [Asn'. Valq-ANG II or 2.5 mg. kg bw-' CS-EXT.

9) Cardiovascular regulation in A. rostrata may be mediated through two subtypes of

ANG II receptors; an AT,-like, Losartan sensitive subtype and an ATJike, PD123319

sensitive subtype.

REFERENCES

Akagi Hl Hayashi T, Nakayarna T, Nakajima T, Watanabe TX 8 Sokabe H 1992

Comparative studies on ang iotensin. Chemical Phamaceutical Bulletin 30 2498-

2502.

Antonaccio MJ 1982 fnhibitors of the renin-angiotensin system as new antihypertensive

agents. Clinical and Experimenfal Hyperpettensin Part A Theory and Practice

A4 (1 -2) 2746.

Aoyagi Tl Kunimoto S. Morishima Hl Tekeuchi T & Umezawa H 1971 Effect of pepstatin

on acid proteases. Journal of Antibiotics 24 687-694.

Arillo A, Uva B & Vallarino M 1981 Renin activity in rainbow trout (Saïmo gairdnen

Rich.) and effects of environmental ammonia. Compamtive Biochemistry and

Physiology 68A 307-3 1 1.

Armour KJ, O'Toole LB & Hazon N 1993 Mechanisms of ACTH- and angiotensin II-

stimulated 1 - u -hydroxycorticosterune secretion in the dogfish, Scyliorhinus

canicula . Journal of Molecuïar Endocrinoiogy 1 O 23 5-244.

Arnold-Reed DE 8 Balment RJ 1994 Peptide hormones influence in vitro interrenal

secretion of cortisol in the trout. Oncorhynchus mykr'ss- General and

Comparative Endocrinology 96 85-9 1 .

Audigé J 191 0 Contribution à l'étude des reins des poissons téléostéens. Archives de

Zoologie Expérimentale et Générale 4 275-625.

Bailey JR 8 Fenwick JC 1975 The effect of stanniectomy and plasma clacium levels on

blood pressure in the eel. Anguilla rostrata Lesueur. Comparative Biochemistry

and Physiology 51 693-697.

Bailey JR 8 Randall DJ 1981 Renal perfusion pressure and renin secretion in the

rain bow trout. Salmo gairdnen. Canadian Journal of Zoology 59 1 220-1 226.

Balment RJ & Carrick S 1985 Endogenous renin-angiotensin system and drinking

behaviour in flounder. Amencan Journal of Physiology 248 R I 57-R160.

Bauchot R 1953 Anatomie comparée des corpuscles de Stannius chez les

Téléostéens. Archives de Zoologie Expérimentale et Générale 89 1 47-1 69.

Bean JW 1942 Specificity in the renin-angiotensinogen reaction. Amencan Journal of

Physiology 1 36 732-742.

Beldent V, Michaud A, Wei L, Chauvet M-T 8 Corvol P 1993 Proteolytic release of

human angiotenisin-converting enzyme. Joumal of Biological Chemistry 268

26428-26434.

Belsare DK 1973a Comparative anatomy and histology of the corpuscles of Stannius in

teleosts. Zeitschrift für Mikmskopisch-Anatomische Forschung (Leipzig) 87 445-

456,

Belsare DK 19736 Histogenesis of the interrenal corpuscles. the chromafin tissue and

the corpuscles of Stannius in Channa punctatus Bloch. (Actinopyerygii.

Percomorphi). Ce11 and Tissue Research 6 343-349.

Bergsrna DJ. Ellis Cl Nuthulaganti PR. Nambi P. Scaife K. Kumar C & Aiyar N 1993

Isolation and expression of a novel angiotensin II receptor from Xenopus laevis

hea rt. Molecular Phamacology 44 277-284.

Bhattacharya TK 8 Butler DG 1978 Fine structure of the corpuscles of Stannius in the

toadfish. Journal of Morphology 155 27 1-286.

Blair-West JR, Coghlan JP. Denton DA, Gibson AP. Oddie CJ. Sawyer WH 8 Scoggins

BA 1977 Plasma renin activity and blood corticosteroids in the Australian

lu ngfish. Neoceratodus forsten. Journal of Endocnnology 74 1 37-1 42.

Borriraja V. Henderson IW & Chester-Jones 1 1973 Renal fractions affeding the

concentration of plasma cortisol in the eel, Anguilla anguilla L. Journal of

Endocrhology 57 1 3-1 7.

Boyd GW 1977 An inactive higher-molecular-weight renin in normal subjects and

hypertensive patients. Lancet 1 21 5-21 8.

Brown AJ, Clark SA & Lever AF 1983 Slow rise and diumaf changes of blood pressure

with saralasin and angiotensin I I in rats- American Journal of Physiology 244

F84-F88.

BuckIey JP 1972 Actions of angiotensin on the central nervous system. Federation

Proceedings 31 1332-1 337.

Bumpus FM. Catt KJ. Chiu AT, DeGasparo M. Goodfriend Tl Husain A. Peach MJ,

Taylor DG 8 Timmermans, PBMWM 1991 Nomenclature for angiotensin

receptors. Hyperfension 17 270-723.

Burton J. Poulsen K 8 Haber E 1975 Cornpetitive inhibitors of renin: inhibitors effective

at physiological pH. Biochemistry 14 3892-3898.

Butler DG & Cadinouche AMZ 1995 Fractional reabsorption of calcium, rnagnesiurn and

phosphate in the kidneys of freshwater North American eels (Anguilla rostrata

Lesueur) following removal of the corpuscles of Stannius. British Journal of

Comparative Physiology 165 348-358.

Butler DG 8 Oudit GY 1995 Corpuscle of Stannius and blood flow regulation in

freshwater North American eels. Anguilla rostrata Lesueur. Joumal of

Endocrinology 145 181 -1 94.

Butler DG. Oudit GY & Cadinouche AZM 1995 Angiotensin 1-and Il-and norepinephrine-

mediated pressor responses in an ancient holostean fish. the bowfin (Amia

calva). General and Comparative Endocnnology 98 289-302.

Butler DG, ZandevakiIi R 8 Oudit GY 1998 Effect of ANG II and III and angiotensin

receptor blockers on nasal salt gland secretion and arterial blood pressure in

conscious Pekin ducks (Anas platyrhynchos). Joumal of Comparative Physiology

and Biochemistry i 68 21 3-224.

Campbell DJ, Bouhnik J. Coexy E, Menad J & Corvol P 1985 Characterization of

precursor and secreted forms of human angiotensinogen. Joumal of Clinical

Investigation 75 1880-1 893.

Capelli JP, Wesson LG 8 Aponte GE 1970 A phylogenetic study of the renin-

angiotensin system. American Journal of Physiology 21 8 1 171 -1 178.

Capreol SV & Sutheriand LE 1968 Comparative morphology of juxtaglomenilar cells. 1.

Juxtag lomerular cells in fish. Canadian Journal of Zoology 46 249-256.

Carpenter SJ 8 Heyl HL 1974 Fine structure of the corpuscles of Stannius of Atlantic

salrnon during freshwater spawning joumey. General and Comparative

Endocrinology 23 2 1 2-223.

Carrick S & Balment RJ 1983 The renin-angiotensin system and dnnking in the

eu ry ha1 ine lounder, Platichthys flesus. General and Comparative Endocnnology

51 423433.

Carroll RG & Opdyke DF 1982 Evolution of angiotenisn il-induced catecholamine

release. American Journal of Physiology 243 R65-R69.

Chang RSL 8 Lott VJ 1990 Two distinct angiotensin I I receptor binding sites in rat

adrenal revealed by new selective nonpeptide ligands. Molecular Phamacology

29 347-351.

Chester-Jones Il Henderson IW, Chan DKO, Rannklin JC, Mosley W. Brown JJ, Lever

AF. Robertson JIS & Tree M 1966 Pressor activity in extracts of the corpuscles

of Stannius from the European eel (Anguilla anguilla L.). Joumal of

Endocrinology 34 292408.

Chiu AT, Herblin WF. Ardecky RJ. McCall DE. Carini DJ. Duncia JV. Pease LJ Wexler

RR. Wong PC. Johnson AL & Timmermans PBMWM 1989 Identification of

ang iotensin I I receptor su btypes. Biochemical and Biophysical Research

Communications 165 1 96-203.

Chiu AT, McCall DE, Price WA, Wong PC and Carini DJ 1990 Nonpeptide angiotensin

II receptors antagonists VU. Cellular and biochemical phamiacology of DuP 753.

an orally active antihypertensive agent. Joumal of Phannacology and

Experimental Therapeutics 252 71 1-71 8.

Churchill PC, Malvin RL, Churchill MC 8 McDonald FD 1979 Renal fucntion in Lophius

americanus: Effects of angiotensin I 1. Amencan Journal of Physiology 236 R297-

R301.

Cobb CS & Brown JA 1992 Angiotensin II binding to tissues of the rainbow trout. Salmo

gairdneri, studies by autoradiography. Joumal of Comparative Physiology 162

1 97-202.

Cohen RS. Pang PKT 8 Clark NB 1975 Ultrastructure of the Stannius corpuscles of the

killifish, Fundulus heteroclitus. and its relation to calcium regulation. General and

Comparative Endocrinology 27 41 3-423.

Davis JO, Freeman RH, Johnson JA 8 Spielman WS 1974 Agents which block the

actions of the renin-angiotensin system- Circulation Research 34 279-285.

De Gasparo M. Levens NR. Kamber B. Furet P. Whitehead S. Brechler V 8 Bottari SP

1993 The Angiotenisn II AT, Receptor Subtype. In Angiotensin receptor, pp 95-

118. Eds JM Saavedra 8 Timmermans PBMWM. New York: Plenum Press.

De Smet D 1962 Considerations on the Stannius corpuscles and intemenal tissue of

bony fishes. especially based on researches into Amia calva. Acta Zoologica

(Stockholm) 43 20 1 -2 1 9.

Dudley DT. Panek RL. Major TC. Lu GH. Bruns RF. Klinkefus BA, Hodges JC 8

Weishaar RE 1990 Subclasses of angiotensin II binding sites and their functional

sig nificance. Molecular Phamacology 38 370-377.

Dunn RS & Perks AM 1975 Comparative studies of plasma kinins: The kailikrein-kinin

system in poikilothem and other vertebrates. General and Comparative

Endocrinology 26 165-1 78.

Dzau VJ. But DW 8 Pratt RE 1988 Molecular biology of the renin-angiotensin system.

American Journal of Physiology 255 F563-F573.

Edwards JG 1940 The vascular pole of the glomenilus in the kidney of vertebrates. The

Anatomieal Record 76 381 -389.

Ehlers MRW & Riordan JF 1989 Angiotensintonverting enzyme: New concepts

conceming its biological role. Biochemistry 28 83 1 1 -53 1 8.

Ehlers MRW 8 Riordan JF 1990 Angiotensin-converting enzyme. Biochemistry and

molecular biology. In Hypertension: Pathophysiology, Diagnosis, and

Management, pp.1217-1231. Eds LH Laragh & BM Brenner. New York: Raven

Press.

Ehlers MRW 8 Riordan JF 1991 Angiotensin-converting enzyme. Zinc- and inhibitor-

binding stoichiometries of the somatic and testis isozymes. Biochemist~ 30

71 18-7126,

Erdos EG 1976 Conversion of angiotensin I to angiotensin II. Amencan Journal of

Erdos EG 1977 The angiotensin I converting enzyme. Federafion Proceedings 36

1760-1 765.

Erdos EG 1990 Angiotensin I converting enzyme and the changes in our concepts

throug h the years. Hypertension 16 363-367.

Erdos EG 8 Skidgel RA 1987 The angiotensin 1-converting enzyme. Laboratoq

Investigation 56 345-348.

Evin G. Gardes J, Kreft C, Castro 8, Corvol P & Menard J 1978 Soluble pepstatins: a

new approach to blockade in vivo of the renin-angiotensin system. Clinical

Science and Molecular Medicine 55 1673-1 695

Fernley RT. John M. Niall HD & Coghlan JP 1986 Purification and characterization of

ovine angiotensinogen. European Journal of Biochemistry 154 597-601.

Ferguson RK. Brunner HR & Turini GA 1977 A specific orally active inhibitor of

angiotensin converting enzyme in humans. Lancet 1 75-779.

Ford P 1959. Some observations on the corpuscles of Stannius. In Comparative

Endocrinology. pp. 728-734. Ed A Gorbman. New York: Wiley.

Fujita H 8 Honma Y 1967 On the fine structure of corpuscles of Stannius of the eel.

Anguilla japonica. Zeitschrift für Zellforschung und Mikroskopische Anatomie 77

175-1 87.

Galli-Phillips SM 1991 Evidence for the presence of an active angiotensin system in the

nurse s hark. Gynchlymostoma cinatum. In Book of Abstracts. 9 International

Symposium on Fish Physiology pp. 25.

Garrett FD 1942 The development and phylogeny of the corpuscles of Stannius in

ganoid and teleostean fishes- Journal of Morphology 70 41 -67.

Gauten D, Paul M, Deboden A. Unger TH & Lang RE 1984 The brain renin angiotensin

systern in central cardiovascular control. Contributions to Nephrology 43 1 14-

128.

Gavaras H. Brunner HR & Turini GA 1978 Antihypertensive effect of the oral converting

enzyme inhibitor SQ 14225 in humans. New England Journal of Medicine 298

991 -995.

Goodfriend TL, Furhquist F. Gutmann F, Knych E. Hollemans Hl Allrnann D. Kent K 8

Cooper T 1972 Clinical and conceptual use of angiotensin recepton. pp. 549-

563. In Hypedension '72. Eds J Genest & E Kiow. New York: Springer Verlag.

Gray DA. Gerstberger R & Simon E 1989 Role of angiotensin II in aldosterone

regulation in the Pekin duck. Journal of Endocnnology 123 445-452.

Griendling KK, Lassique B. Murphy TJ & Alexander RW 1994 Angiotensin II receptor

p harmacolog y. Advances in Pharmacology 28 269-306.

Grill G, Granger P 8 Thourau K 1972 The renin angiotensin systern of amphibians. 1.

Determination of the renin content of arnphibian kidneys. Pflugers Archiv;

European Journal of Physiology 331 1-12.

Gross F, Lazar J & Orth H 1972 Inhibition of the renin-angiotensin reaction by

pepstatin. Science 175 656.

Haber E 1985 WiII renin inhibitors influence decision-making in antihypertensive

therapy. Journal of Hypertension 3 (Suppl. 2 ) S71-80.

Hackenthal E. Hackenthal R 8 Hilgenfeld U 1978 Isorenin, pseudorenin. cathepsin D

and renin. A comparative enzymatic study of angotensin-forming enzymes.

Biochimica et Biophysica Acta 522 574-588.

Hall JE & Brands MW 1992 The renin-angiotensin-alsosterone system: Renal

mechanisms and circulatory homeostasis. In The Kidney: Physiology and

Pathophysiology, pp. 1455-1 504. Eds DW Seldin & G Giebisch. New York:

Raven Press.

Hall MM. Khosla MC. Khairallah PA & Burnpus FM 1974 Angiotensin analogs: the

influence of sarcosine substituted in position 1. Journal of Phamacology and

Expeninental Therapeutics 158 222-228.

Harper RA 8 Stephens GA 1985 Blockade of the pressor response to angiotensin I and

II in the bullfrog. Rana catesbeiana. General and Comparative Endocnnology 60

227-235.

Hasegawa Y. Cipolle M. Watanabe TX. Nakajima T, Sokabe H 8 Zehr JE 1984a

Chemicai structure of angiotensin in the turtle. Pseudemys scripta. General and

Comparative Endocnnology 53 1 59-1 62.

Hasegawa K. Nishimura H & Khosla MC 1993 Angiotensin-induced endothelium-

dependent relaxation of fowl aorta. Amencan Journal of Physiology 264 R903-

911.

Hasegawa Y. Nakajirna T & Sokabe H 1983a Chemical structure of angiotensin formed

wih kidney renin in the Japanese eel, Anguilla japonica. Biomedical Research 4

41 7-420.

Hasegawa Y, Watanabe TX. Nakajima T B Sokabe H 19846 Chemical structure of

angiotensin formed by incubating plasma with corpuscle of Stannius in the

Ja panese goosefish . Lophius litulon. General and Comparative Endocnnology

54 264-269.

Hasegawa Y. Watanabe TX. Sokabe H 8 Nakajima T 19836 Chemical structure of

angiotensin in the bullfrog. Rana catesbeiana. General and Comparative

Endocrinology 50 75-80.

Hayashi T. Nakayarna T. Nakajima T 8 Sokabe H 1978 Comparative studies on

angiotensins. V. Structure of angiotensin fomed by the kidney of Japanese

goosefish and its identification by dansyl method. Chemical and Phamaceutical

Bulletin 26 2 1 5-2 1 9.

Hazon N. Balment RJ. Perrott M 8 O'Toole LB 1989 The renin-angiotensin system and

vascular and dipsogenic regulation in elasmobranches. General and

Comparative Endocrinology 74 230-236.

Hazon N & Henderson IW 1985 Factors affecting secretory dynamics of l-alpha-

h ydroxycorticosterone in the dogfish. Scyliorhinus canicula. General and


Henderson IW. Jotisankasa V. Mosely W 8 Oguri M 1976 Endocrine and environmental

influences upon plasma cortisol concentrations and plasma renin activity of the

eel, anguilla anguilla L. Journal of Endocrinology 70 8 1 -95.

Henderson IW, Oliver JA, McKeever A 8 Hazon N 1981 Phylogenetic aspects of the

ren in-ang iotensin system. I n Advances in Animal and Comparative Physiology,

pp. 355-363. Eds G Pethes 8 VL Freny. Oxford: Pergamon Press.

Herblin WF. Chiu AT, McCall DE. Ardecky RJ & Canni DJ 1991 Angiotensin II receptor

heterogeneity. A m e m n Journal of Hypertension 4 299s-302s.

Heyl HL 1970 Changes in the corpuscles of Stannius during the spawning joumey of

a tian tic salmon (Salmo salar). General and Comparative Endocnnology 1 4 43-

52.

Hidaka H, Sawada SI Sato R 8 Oka H 1985 An improved method for measuring

angiotensin I converting enzyme activity using a highly sensitive angiotensin II

radioimmunoassay. Endocnnologia Japonica 32 803-809.

H irano T 1 989 The corpuscle of Stannius. In Vertebrate Endocrinology: Fundamentals

and Biomedical lrnplications Vol 3. pp. 139-1 62. Eds. PKT Pang & MP

Schreibman. San Diego: Academic Press Inc.

Hirano T 8 Hasegawa S 1984 Effects of angiotensins and other vasoactive substances

on drinking in the eel, Anguilla japonica. Zoological Science 1 106-1 13.

Hirano T. Takei Y & Kobayashi H 1978 Angiotensin and drinking in the eel and the frog.

1 n Osmotic and volume regulation, Alfred Benzon Symposium XI, pp. 1 23-1 28 -

Eds C Barker-Jorgensen & E Skadhauge Copenhagen: Munksgaard.

Hirose S 8 Murakami K 1992 Molecular biology of renin. Blood Vessels 2 65-69.

Hooper NM 1990 Angiotensin converting enzyme: implications from molecular biology

for its ph ysiolog ical functions. International Journal of Biochemistry 23 641 -647.

Hulthen 1 & Hokfelt T 1978 The effect of the converting enzyme inhibitor SQ20 881 on

kinins. renin-angiotensin-aldosterone and catecholamines in relatons to blood

pressure in hypertensive patients. Acta Medica Scadinavica 204 497-502.

lkemoto F, Takaori K, lwao H & Yamaoto K 1982 lntrarenal localization of renin binding

substances in rats. Life Sciences 31 101 1-101 6.

l nagarni T 8 Murakami K 1977 Purification of high molecular weight forms of renin from

hog kidney. Circulation Research 41 (Suppl. 2 ) 1 1-1 6.

lnouye A. Kataoka K 8 Tsujioka T 1961 On a kinin-like substance in the nervous tissue

extracts treated with trypsin. Japanese Journal of Physiology 11 31 9-324.

ltskowitz HD 8 McGiff JC 1974 Hormonal regulation of the renal circulation. Circulation

Research 34 (Suppl. 1) 65-73.

Jackson BA. Brown JA. Oliver JA 8 Henderson IW 1977 Actions of angiotensin on

single nephron rate of trout. Salrno gairdnen. adapted to fresh- and sea-water

environ ments. Journal of Endocrinology 75 32P.

Johnson CI 1990 Biochemistry and pharmacology of the renin-angiotensin system.

Dnigs 39 (Suppl. 1) 21-31.

Johnson CI, McGrath %P. Millar JA & Matthews PGF 1979 Long-terni effects of

captopril (SQ14225) on blood pressure and hormone levels in essential

hypertension. Lancet 2 193-1 96.

Johnson DW 1972 Variations in the interrenal and corpuscles of Stannius of Mugi1

cephalus from the Colorado river and its estuary. Generai and Comparative


Johnson JA & Davis JO 1972 Effects of an angiotensin II analog on blood pressure and

adrenal steroid secretion in dogs with thoracic caval constriction (abstract).

Physiology 1 5 1 84.

Johnson JA & Davis JO 1973 EKect of a specific wmpetitive antagonist of angiotensin

II on artenal pressure and adrenal steroid secretion in dogs. Circulation

Research 32 (Suppl. 1) 1 59-1 68.

Kaneko T. Hasegawa S 8 Hirano T 1992 Embryonic origin and development of the

corpuscles of Stannius in churn salmon (Oncorhynchus keta). Cell and Tissue

Research 268 65-70.

Khosla MC. Nishimura H. Hasegawa Y 8 Bumpus FM 1985 Identification and synthesis

of [1 -asparagine. 5-valine. 9-glycine] angiotensin I produced from plasma of

American eel . Anguilla rostrata. General and Comparative Endocnnology 57

223-233.

Khosla MC. Smeby RR 8 Bumpus FM 1974 Structure-activity relationship in

angiotensin II analogs. In Angiotensin, pp. 126-161. Eds IH Page 8 FM

Bumpus. Berlin: Springer-Verlag.

Kloas W & Hanke W 1992 Angiotensin II binding sites in frog kidney and adrenal.

Peptides 1 3 349-354.

Ko bayas hi H & Ta kei Y 1 996a in The Renin-Angiotensin System: Comparative aspects.

Zoophysiology, Vol. 35, pp. 1-9. Berlin: Springer International.

Kobayashi H 8 Takei Y 19966 In The Renin-Angiotensin System: Comparative aspects.

Zoophysiology, Vol. 35, pp. 77-88. Berlin: Springer International-

Kohler G & Milstein C 1975 Continuous cultures of fused cells producing antibody of

predefined specificity. Nature 256 495-497. .

Krishnamurthy VG 1 967 Development of the corpuscles of Stannius in the anabantid

teleost, Colisa lalia. Journal of morphology 123 109-1 20.

Krishnamurthy VG 1976 Cytophysiology of corpuscles of Stannius. International Review

of Cytology 46 1 77-246.

Krishnamurthy VG & Bern HA 1971 Innervation of the corpuscles of Stannius. General

and Comparative Endocrinology 16 162-1 65.

Krishnamurthy VG 8 Bern HA 1969 Correlative histological study of the corpuscles of

Stannius and the juxtaglornerular cells of teleost fishes. General and

Comparative Endocrinology 13 31 3-335.

Krishnamurthy VG & Bern HA 1973 Juxtaglomenilar cell changes in the euryhaline

freshwater fish, Tilapia mossambica, during adaptation to sea water. Acta

Zoologica 54 9-1 4.

Lacy ER & Reale E 1990 The presence of a juxtaglomerular apparatus in

elasmo branch fish. Anatomy and Embryology 1 82 249-262.

Lafeber FPJG. Flik G. Wendelaar-Bonga SE 8 Perry SF 1988a Hypocalcin from

Stannius corpuscles inhibits gill calcium uptake in trout. Amencan Journal of

Physiology 254 R89 1 -R896.

Lafeber FPJG, Hanssen RGJM, Choy YM. Flik G. Hermann-Eriee MPM, Pang PKT 8

Wendelaar-Bonga SE 1988b identification of hypocalcin (teleocalcin) isolated

from trout corpuscles of Stannius. General and Comparative Endocrinology 69

1 9-30.

Lagois MD 1974 Granular epitheloid (juxtaglomerular) cell and renovascular

morphology of the coelacanth Latirnena chalumnae Smith (Crossopterygii)

compared with that of other fishes. General and Comparative Endocrinology 22

296-307.

Le Noble FAC. Hekking JWM. Van Straaten HWM., Slaaf DW & Struyker-Bourdier HAJ

1991 Angiotensin II stimulates angiogenesis in chorio-allantoic membrane of the

chicken embryo. European Joumal of Phamacology 195 305-306.

Lipke DW. Thomas RL & Olson KR 1987 Characterization of angiotensin-converting

enzyme in the gills of rainbow trout, Salmo gairdnen (Richardson). Fish

Physiology and Biochemistry 3 91 -97.

Lopez E. Tisserand-Jochem E-M. Vidal B. Milet C. Lallier F 8 Maclntyre I 1984 Are

corpuscles of Stannius the parathyroid glands in fish? Immunocytochemical and

u l t rast ructurôl argu ments. 1 n Endocrine control of Bone and Calcium Metabolism,

Vol. 8B. pp. 283-286. Eds DV Cohn, JT Potts & T Fu!Ïta. Amsterdam:

ElsevierINorth-Holland Biomedical Press.

Lumbers ER 1971 Activation of renin in human amniotic fluid by low pH. Enzymologia

40 329-336.

Lund-Johansen P & Omvik P 1984 Long-terrn haemodynamic affects of enalapril (alone

and in combination with hydrochlorothiazide) at rest and during exercise in

essential hypertension. Journal Hypertension 2 (Su ppl. 20) 49-56.

Ma SWY & Copp DH 1978 Purification. properties and action of a glycopeptide fom the

corpuscle of Stannius which affects calcium metabolism in the teleost. In

Comparative Endocnnology. pp 283-286. Eds PJ Gaillard et al. Amsterdam:

Elsevier.

Mandich A 8 Massari A 1994 Presence of angiotensin II immunoreactivity in the ovary

of the rainbow trout, Salmo gaidneri General and Comparative Endocrinology 96

1-5.

Marciniszyn J I Hartsuck JA & Tang J 1976 Mode of inhibition of acid proteases by

pepstatin . Journal of Biological Chemistry 251 7088-7094.

Marra LE. Butler DG. Zhang OH, Oudit GY & Youson JH 1998 Corpuscles of Stannius

and stanniocalcin-like immunoreactivity in the white sucker (Catostomus

commerson~: evidence for a new cell-type. Ce11 and fissue Research 294 1 55-

164.

Marsigliante S. Muscella A. Vilella S. Nicolardi G. Ingrosso L, Ciardo VI Zonno V,

Vinson GP. Ho MM 8 Storelli C 1995 A monoclonal antibody to mammalian

angiotensin II AT, receptor recognizes one of the angiotensin II receptor isoform

expressed by the eel (Anguilla anguilla). Journal of Molecular Endocrinology 16

45-56.

Marsigliante S, Verri TI Barker SI Jimenez E. Vinson GP & Storelli C 1994 Angiotensin

II receptor subtypes in eel (Anguilla anguilla). Journal of Molecular Endocrinology

12 61 -69.

Messerii FH. Weber MA & Bninner HR 1996 Angiotensin II receptor inhibition: A new

therapeutic principle. Archives of Interna1 Medicine 156 1957-1 966.

Milet C. Buscaglia M. Chartier MM. Martelly E & Lopez E 1984. Comparative effect of

Stannius corpuscles extracts and 1-34 hPTH in the Anura, Xenopus laevis.

General and Comparative Endocrinology 53 497.

Miyazaki M. Komori Tl Okunishi H & Toda N 1979 Inhibition of dog renin by pepstatin A.

Japanese Circulation Journal 43 81 8-823.

Moore AF. Strong JH 8 Buckley JP 1981 Cardiovascular actions of angiotensin in the

fowl (Gallus domesticus) I 1. Angiotensin analog agonists and antagonists-

Research Communications in Chernical Pathology and Phamacology 32 447-

457.

Murakami E. Eggena P. Barret JD & Sambhi MP 1984 Heterogeneity of renin substrate

released from hapatocytes and in brain extracts. Life Sciences 34 385-392.

Nakajima T. Nakayama T & Sokabe H 1971 Examination of angiotensin-like substances

from renal and extrarenal sources in mammalian and nonmammalian species.

General and Comparative Endocnnology 1 7 458-466.

Nakarnura Y, Hishimura H 8 Khosla MC 1982 Vasodepressor action of angiotensin in

conscious chickens. American Journal of Physiology 243 H456eH462.

Nakayama T, Nakajima T 8 Sokabe H 1973 Comparative studies on angiotensin. III.

Structure of fowl angiotensin and its identification by DNS-method. Chemical and

Pharmaceutical Bulletin 21 2085-2087.

Nakayama T, Nakajima T & Sokabe H 1977 Comparative studies on angiotensin. IV.

Structure of snake (Elaphe climocophora) angiotensin. Chemical and

Pharrnaceutical Bulletin 25, 3255-3260.

Nishirnura H 198Oa Comparative endocrinology of renin and angiotensin. In The Renin-

Angiotensin System. pp. 29-77. Eds JA Johnson & Anderson RR. New York:

Plenum Press.

Nishimura H 1980b Evolution of the renin-angiotensin system. In Evolution of vertebrate

endocrine systems. Eds PKT Pang 8 A Epple. Graduate Studies, Texas Tech

University 21, 373404.

Nishimura H 1985 Evolution of the renin-angiotensin system and its role in control of

cardiovascular function in fishes. In Evolutionary Biology of Primitive Fishes, pp

275-293. Eds RE Foreman, A Gorbman, JM Dodd, R Olsson. New York:

Plenum Press.

Nishimura H & Qin Z 1994 Control of renin release in teleosts. (abstract) Physiology 37

A34.

Nishimura H & Sawyer WH 1976 Vasopressor. diuretic, and natriuretic responses to

angiotensins by the Amencan eel. Anguilla rostrata. General and Comparative


Nishimura Hl Norton VM & Bumpus FM : 978 Lack of specific inhibition of angiotensin II

in eels by angiotensin antagonsts. American Journal of Physiology 235 H95-103.

Nishimura H & Ogawa M 1973 The renin-angiotensin systern in fishes. Amencan

Z0010gist i 3 823-838.

Nishimura Hl Ogawa M 8 Sawyer WH 1973 Renin-angiotensin system in primitive bony

fishes and a holocephalian. Amencan Journal of Physiology 224 950-956.

Nishimura H, Oguri M. Ogawa M. Sokabe H 8 Imai M 1970 Absence of renin in kidneys

of elasmobranches and cyclostomes. American Journal of Physiology 218 91 1-

91 5.

OIToole LB, Armour KJ, Decourt Cl Hazon N, Lahlou B 81 Henderson IW 1990

Secretory patterns of 1 -alpha-hydroxycorticosterone in the isolated perfused

interrenal gland of dogfish, Scyrhiolinus canicula. Journal of Molecular

Endocnnology 5 55-60.

Ogawa M 1968 Osmotic and ionic regulation in goldfish following removal of the

corpuscles of Stannius or the pitutary gland. Canadian Journal of Zoology 46

669-676.

Ogawa M 1977 Evolution of juxtaglomerular apparatus. Gunma Symposium on

Endocrinology 14 75-8 1 .

Ogawa M & Sokabe H 1982 Hypocalcemic effect of homologous angiotensin-like

substances produced by the renin-like enzyme in the corpuscle of Stannius in

the Japa nese eel. Anguilla japonica. General and Comparative Endocnnology 47

36-41.

Ogawa M 8 Oguri M 1978 Occurrence of the renin-angiotensin system in the

vertebrates. Japanese Head Journal 19 791 -798.

Ogawa M. Oguri M. Sokabe H & Nishimura H 1972 Juxtaglomenilar apparatus in the

verte brates. General and Comparative Endocnnology Suppl. 3 , 374-38 1 .

Oguri M 1966 Electron-microscopic observtions on the corpuscles of Stannius in

goldfis h . Bulletin of the Japanese Society of Scientific Fisheries 32 903-908.

Oguri M 1978 Presence of juxtaglomenilar cells in the holocephalian kidney. General

and Comparative Endocnnology 26 170-1 73.

Ogun M. Ogawa M & Sokabe H 1970 Absence of juxtaglomenilar cells in the kidneys of

ch rond richth yes and Cyclstorni. Bulletin of the Japanese Society of Scientific

Fishenes 36 881 -884.

Oguri M & Sokabe H 1968 Juxtaglomenilar cells in the teleost kidneys. Bulletin of the

Japanese Society of Scientific Fisheries 34. 882-888.

Olson KR 1992 Blood and extracellular fluid volume regulation. In Fish Physiology, The

Cardiovascular system Vol. XI18 . pp. 135-254. Eds WS Hoar. DJ Randal 8 AP

Farrell, New York: Academic Press.

Olson KR. Conklin DJ. Weaver L Jr. Duff DW, Heman CA. Wang X 8 Conlon JR 1997

Cardiovascular effects of homologous bradykinin in rainbow trout. Amencan

Journal of Physiology 272 RI1 12-1 1 120.

Olson KR. Lipke DW. Kullman D, Evan AP 8 Ryan JW 1989 Localization of

angiotensin-conveiting enzyme in the trout gill. Joumal of Expenmental Zoology

250 109-1 15.

Ondetti MA & Cushman DW 1982 Enzymes of the reninangiotensin system and their

in hibitors. Annual Review of Biochemistry 51 283-308.

Ondetti MA. Rubin B 8 Cushman DW 1977 Design of specific inhibitors of angiotensin

converting enzyme: New class of orally active antihypertensive agents. Science

196 441-443.

Opdyke DF 8 Holcombe H 1976 Response to angiotensin I and II and to the Al-

converting enzyme inhibitor in a shark. Amentan Journal of Physiology 231

1750-1 753.

Oudit GY 8 Butler DG 1995 Angiotensin II and cardiovascular regulation in a freshwater

teleost. Anguilla rostrata Lesueur. Amencan Journal of Physiology 269 R726-

735.

Panthier JJ. Foot S. Chambreaud B. Strosberg AD. Corvol P & Rougeon F 1982

Complete amino acid sequence and maturation of mouse submaxillary gland

renin precursor. Nature 298 90-92.

Parsons JA. Gray D. Rafferty B 8 Zanelly JM 1978 Evidence for a hypercalcemic factor

in the fish pituitary. imrnunologically related to mammalian parathyroid hormone.

In Endocrinology of Calcium Metabolism, pp. 1 1 1-1 14. Eds DH Copp 8 RV

Talmage. Amstedam: Excerpta Medica.

Peach MJ 1 977 Renin-angiotensin system: biochemistry and mechanism of action.

Physiological Re vie ws 57 3 1 3-370.

Peach MJ. Bumpus FM & Khairallah PA 1971 Release of adrenal catecholamines by

angiotensin 1. Joumal of Phamacology and Experimental Therapeutics 176 366-

376.

Perrott MN & Balment RJ 1990 The renin-angiotensin system and the regulation of

plasma cortisol in the flounder. Platichtyys fiesus. General and Comparative

Endocrinology 78 41 4420.

Perrott MN 8 Balrnent RJ 1985 Drinking behavior and the renin angiotensin system in .

euryhaline and stenohaline fish (abstract). Journal of Endocrinology 107 193.

Perroît MN, Grierson CE, Hazon N & Balment RJ 1992 Dn'nking behavior in sea water

and fresh water teleosts, the rote of the renin-angiotensin system. Fish

Physiology and Biochemistry 1 O 1 6 1 -1 68.

Polanco MJ. Mata M 1, Agapito MT 8 Recio JM 1 990 Angiotensin-converting enzyme

distribution and hypoxia response in mamrnals, bird, and fish. General and


Poulsen K. Burton J & Haber E 1973 Competitive inhibitors of renin. Biochemistry 12

3877-3882.

Rasquin P 1956 Cytoogical evidence for a role of the corpuscles of Stannius in the

osrnoregulation of teleosts. The Biological Bulletin 1 1 1 399-409.

Rotmensch HH, Vlasses PH and Ferguson RK 1988 Angiotensin-converting enzyme

in hi bitors- Medical Clinics of North Amenca 72 399-425.

Rowe BP. Grove KL. Sayler DL 8 Speth RC 1991 Discrimination of angiotensin II

receptor subtypes in the rat brain using nonpeptidic receptor antagonists.

Regulatory Peptides 33 45-53.

Ryan GB, Alcorn D. Coghlan JP 8 Scoggins BA 1979 The granulated peripolar

epithelial ceIl: a potential secretory component of the renal juxtaglomenilar

cornplex. Nature 277 655-656.

Salzet M. Verger-Bocquet M. Wattez C 8 Malecha J 1992 Evidence for angiotensin-like

molecules in the central nervous system of the leech Theromyzon tessulatum

(O. F. M. ). A possible diuretic effect. Comparative Biochemistry and Physiology

1O1A 83-90.

Salzet M, Wattez C, Baert J-L & Malecha J 1993 Biochernical evidence of angiotensin

Il-like peptide and proteins in the brain of the'rhynchobdellid leech Theromyzon

tessula tum. Brain Research 631 247-255.

Sandberg K. Bar M. Ji Hl Markorck A. Millan MA 8 Catt KI 1990 Angiotensin Il-induced

calcium rnobilization in oocytes by signal transfer through gap junctions. Science

249 298-30 1.

Sand berg K. Ji H & Millan MA 1992 Amphibian myocardial angiotensin II receptors are

distinct from marnrnalina AT, and AT, receptor subtypes. FEBS Letters 284 281-

283.

Sandor T, Vinso GP, Chester-Jones Il Henderson IW & Whitehouse BJ f 966

Biogenesis of corticosteroids in the European eel (anguilla anguilla L.). Journal of

Endocrinology 34 105-1 15.

Saruta 1, Cook R & Kaplan NM 1972 Adrencortical steroidogenesis: studies on the

mechanism of action of angiotensin and electrolytes. Journal of Clinical

Investigation 51 2239-2245.

Schaffenburg CA. Haas E 8 Goldblatt H 1960 Concentration of renin in kidneys and

angiotensinogen in serum of various species. Amencan Journal of Physiology

199 788-792.

Schiffrin EL 8 Genest J 1983 Tonin-angiotensin II system. In Hypertension. pp. 309-

319. Eds J Genest, O Kuchel, P Hamet 8 M Cantin. New York: McGraw-Hill.

Sen S. Smerby RR 8 Bumpus FM 1969a Antihypertensive effect of an isolated

phospholipid. American Joumal of Physiology 214 337-341.

Sen S. Smerby RR & Bumpus FM 1969b Plasma renin activity in hypertensive rats after

treatment with renin preinhibitor. Amencan Joumal of Physiology 21 6 499-503.

Shoumura S. lwasaki Y. lshicaki NI Ernura S. Hayashi K. Yamahira T, Shoumura K 8

Isono H 1983 Origin of autonornic fibres innervating the parathyroid gland in the

ra bbit, Acta Anatomica 1 1 5 289-295.

Smith RD. Chiu AT. Wong PC, Herblin WF 8 Timmermans PBMWM 1992

Pharmacology of nonpeptide angiotensin II receptor antagonists. Annual Review

of Pharmacology and Toxicology 32 135-1 65.

So YP 8 Fenwick JC 1977 Relationship between net '%lciurn influx across a perfused

isolated eel giH and the development of post-stanniectomy hypercalcemia.

Journal of Experimental Zoology 200 259-264.

So YP & Fenwick JC 1979 The in vivo and in vitro effects of Stannius corpuscles extract

on the branchial uptake of in stanniectomized North American eel (anguilla

rostrata). General and Comparative Endocrinology 37 143-149.

Sokabe H. Nishimura H. Ogawa M 8 Oguri M 1970 Determination of renin in the

corpuscles of Stannius of the teleost. General and Comparative Endocnnology

14 510-516.

Sokabe H 8 Ogawa M 1974 Comparative studies of the juxtaglomerular apparatus.

International Review of Cytdogy 37 270-327.

Sokabe H. Ogawa M. Oguri M 8 Nishimura H 1969 Evolution of the juxtagtomerular

apparatus in the vertebrate kidneys. Texas Reports of Biology and Medicine 27

867-885.

Sokabe H. Oide H, Ogawa M & Utida S 1973 Plasma renin activity in Japanese eels

(anguilla japonica) adapted to seawater or in dehydration. General and

Comparative Endocnnology 21 1 60-1 67.

Swanson LW. Marshall GR. Needleman P & Sharpe LG 1973 Characterization of

central angiotensin II receptors involved in elicitation of drinking in the rat. Brain

Research 49 44 1 -446.

Swanip K & Ahmad N 1978 Studies of corpuscles of Stannius of Notopterus notopterus

in relation to calcium and sodium rich environments. National Academy of

Science Lefters USA 1.239-240.

Szelke M. Leckie BJ. HaHett A. Jones OM, Sueiras J, Atrash B & Lever AF 1982 Potent

new inhibitors of human renin. Nature 299 555-557.

Szelke M. Leckie BJ, Tree M. Brown A. Grant J. Hallett A, Hughes M. Jones DM &

Lever AF 1982 H-77: a potent new renin inhibitor. In vitro and in vivo studies.

Hypertension 4 (Suppl. 2) 59-69.

Takei Y. Hasegawa Y. Watanabe TX. Nakajima K & Hazon N 1993a New angiotensin 1

isolated from a reptile. Alligator mississippiensis. General and Comparative

Endocrinology 90 21 4-2 19.

Takei Y. Hasegawa Y. Watanabe TX. Nakajima K 8 Hazon N 1993b A novel

angiotensin 1 isolated from an elasmobranch fish. Journal of Endocnnology 139

281 -285.

Takei Y, Hirano T & Kobayashi H 1979 Angiotensin and water intake in the Japanese

ee 1. Anguilla japonica. General and Comparative Endocrinology 38 466-475.

Takei Y. ltahara Y. Butler DG. Watanabe TX & Oudit GY 1998 Tetrapod-type [Asp']

angiotensin 1s present in a holostean fish, Amia calva.Genera1 and Comparative

Endocrinology 110 140-146-

Takei Y, Okubo J 8 Yamaguchi K 1988 Effects of cellular dehydration on drinking and

plasma angiotensin II level in the eel, Anguilla japonica. Zoological Science 5 43-

51.

Takemoto Y, Nakajima T. Hasegawa Y, Watanabe TX, Kumagae H 8 Sakakibara S

198. Chemical structure of angiotensin fomed by incubating plasma with the

kidney and the corpuscles of Stannius in the chum salmon, Oncorhynchus keta.

General and Comparative Endocrinology 51 2 19-227.

Tewksbury DA, Dart RA & Travis J 1981 The amino terminal amino acid sequence of

huma n ang iotensinogen. Biochemical and Biophysical Research

Cornmunicafions 99 1 31 1 -1 3 1 5.

i'immermans PBMWM. Benfield P. Chiu AT. Herblin WF. Wong PC & Smith RD *

Angiotensin II receptors and functional correlates. Amencan Journal of

Hyperfension 5 221 5-2355.

Timmermans PBMWM, Wong PC, Chiu AT. Herblin WF. Benfield P, Carini DJ. Lee RJ,

Wexler RR, Saye J-AM & Smith RD 1993 Angiotensin II receptors and

angiotensin II receptor antagonists. Phamacological Review 45 205251.

Turker RK, Hall MM, Yamamoto M. Sweet CS & Bumpus FM 1972 A new, long-lasting

competitive inhibitor of angiotensin- Science 177 1203-1 204-

Uemura H, Tezuka Y. Hasegawa C & Kobayashi H 1994 1mmunohistochemical

investigation of neuropeptides in the central nervous system of the amphioxus,

Branchiostoma belchen Ce11 and Tissue Research 277 279-287.

Umezawa H, Aoyagi T , Morishima H, Matsuzaki M. Hamada M & Tekeuchi T 1970

Pepstatin, a new pepsin inhibitor produced by actinomycetes. Journal of

Antibiotics 23 259-262.

Unsicker K, Plonius T, Lindmar R, Lffeleholz K & Wolf U 1977 Catecholamines and 5-

hydroxytryptamine in corpuscles of Stannius of the salmonid, Salmo indeus L. A

study correlating electron microscopical, histochemical and chemical findings.

General and Comparative Endocnnology 31 121-132.

Urata H, Kinoshinto A, Misono KS. Bumpus FM & Husain A 1990 Identification of a

highly specific chymase as the major angotensin-Il foming enzyme in the heart.

Journal of Biological Chemistry 265 22348-22357.

Vanhoutte PM 1989 Endothelium and control of vascular function: State of the art

lecture. Hypertension 13 658-667.

Verger-Bocquet M. Wattez C, Salzet M & Malecha J 1992 lmmunocytochemical

identification of peptidergic Neurons in cornpartment 4 of the supraesophageal

ganglion of the leech Theromyzon tessulatum (O.F.M.). Canadian Journal of

Zoology 70 856-865-

Viswanathan M. Tsutsumi K. Correa FM 8 Saavedra JM 1991 Changes in expression

of angiotensin receptor subtypes in the rat aorta during development.

Biochemical and Biophysical Research Communications 179 1361 -1 367.

Wagner GF. Fargher RC. Milliken Cl McKeown BA 8 Copp DH 1993 The gill calcium

transport cycle in rainbow trout is correlated with plasma levels of bioactive. not

immunoreactive. stanniocalcin. Molecular and Cellular Endocrinology 93 185-

191.

Wagner GF. Fenwick JC, Milliken C, Park CM. Copp DH & Friesen HG 1988

Comparative biochemistry and physiology of teleocalcin from sockeye and coho

sa1 mon. General and Comparative Endocniology 72 237-246.

Wendelaar-Bonga SE & Greven JAA 1975 A second cell type in Stannius bodies of two

euryhaline teieost species. Cell and Tissue Research 159 287-290.

Wendelaar-Bonga SE. Greven JAA 8 Veenhuis M 1977 Vascularization, innervation,

and ultrastructure of the endocrine cell types of Stannius corpuscles in the

teleost. Gasterosteus aculeatus. Journal of Morphology 153 225-244.

West NH 1989 Methods for measuring blood flow and distribution in intennittently

ventilating and diving vertebrates. In Techniques in comparative Respiratory

Physiolgy-An expenmental approach, pp. 171-1 93. Eds CR Bridges & PJ Butler.

Cambridge: Cambridge Press.

Whitebread S, Mele M, Kamber B & DeGasparo M 1989 Preliminary biochemical

ch tacteriration of two angiotensin II receptor su btypes. Biochemical and

Biophysical Research Communications 163 284-291.

Wilson JX 1984 Coevolution of the renin-angiotensin system and the nervous control of

blood circulation. Canadian Journal of Zoology 62 137-1 47.

Wong PC. Hart SD. Duncia JV 8 Timmermans PBMWM 1991 Nonpeptide angiotensin

II receptor antagonists. XIII. Studies with DuP 753 and EXP3174 in dogs.

European Journal of Pharmacology 202 323-330.

Wong PC. Hart SD. Zaspel AM, Ciu AT. Ardecky RJ, Smith RD & Timmermans

PBMWM 1990 F unctional studies of nonpeptide angiotensin II receptor subtype-

specific ligands: DuP 732 (AH-1 ) and PD1 23177 (AH-2). Journal of

Pharmacology and Experimental Therapeutics 255 584-592.

Wyllie AH. Kerr. J FR 8 Currie AR 1980 Cell death: The significance of apoptosis.

International Review of Cytology 68 25 1 -306.

Yamada C & Kobayashi H (1987). Immunoreactive angiotensin II in the corpuscle of

Stannius of the rainbow trout. Salmo gairdnen. Zoological Science 4 387-390.

Yamamoto M. Turker RK. Khairallah PA & Bumpus FM 1972 Potent cornpetitive

antagonist of angiotensin II. European Journal of Pharmacology 18 31 6-322.

Youson JH 8 Butler DG 1976 Fine structure of the adrenocortical homolog and the

corpuscles of Stannius of Amia calva L. Acta Zoologica (Stockholm) 57. 21 7-230.

Youson JH, Butler DG 8 Chan ATC 1976 Identification and distribution of the

adrenocortical homolog, chromaffin tissue and corpuscles of Stannius in amia

calva L. General and Comparative Endocrinology 29 198-21 1.

Zehr JE. Standen DJ 8 Cipolle MD 1981 Characterization of angiotensin pressor

responses in the turtle. Pseudemys scdpta. Amencan Journal Physiology 240

R276-R281.

Zuker A & Nishimura H 1981 Renal responses to vasoactive hormones in the

ag lomerular toadfis h. Opsanus tau. General and Comparative Endocrinology 43

1-9.

Date post:	15-Mar-2023
Category:	Documents
Upload:	khangminh22
View:	0 times
Download:	0 times

The corpuscles of Stannius contain essential elements of the ...

Documents